Aberrantly methylated genes as markers of breast malignancy

ABSTRACT

The invention is directed to a method of diagnosing a cell proliferative disorder of breast tissue by determining the methylation status of nucleic acids obtained from a subject. Aberrant methylation of several genes including TWIST, HOXA5, NES-1, retinoic acid receptor beta (RARβ), estrogen receptor (ER), cyclin D2, WT-1, 14.3.3 sigma, HIN-1, RASSF1A, and combinations of such genes serve as markers of breast malignancy.

CROSS REFERENCE TO RELATED APPLICATION(S)

This is a continuation-in-part application of U.S. patent applicationSer. No. 09/771,337, filed Jan. 26, 2001, co-pending, which isincorporated herein by reference in its entirety.

GRANT INFORMATION

This invention was made with Government support under P50 CA88843-01,awarded by the National Institutes of Health; and U.S. Public HealthService Grant CA48943. The government may have certain rights in theinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to a method of diagnosing a cellproliferative disorder of breast tissue by determining the DNAmethylation status of nucleic acids obtained a subject.

2. Background Information

Methylation has been shown by several lines of evidence to play a rolein gene activity, cell differentiation, tumorogenesis, X-chromosomeinactivation, genomic imprinting and other major biological processes(Razin, A., H., and Riggs, R. D. eds. in DNA Methylation Biochemistryand Biological Significance, Springer-Verlag, New York, 1984). Ineukaryotic cells, methylation of cytosine residues that are immediately5′ to a guanosine, occurs predominantly in cytosine-guanine (CG) poorregions (Bird, Nature, 321:209, 1986). In contrast, CpG islands remainunmethylated in normal cells, except during X-chromosome inactivation(Migeon, et al., supra) and parental specific imprinting (Li, et al.,Nature, 366:362, 1993) where methylation of 5′ regulatory regions canlead to transcriptional repression. De novo methylation of the Rb genehas been demonstrated in a small fraction of retinoblastomas (Sakai, etal., Am. J. Hum. Genet., 48:880, 1991), and recently, a more detailedanalysis of the VHL gene showed aberrant methylation in a subset ofsporadic renal cell carcinomas (Herman, et al., Proc. Natl. Acad. Sci.,U.S.A., 91:9700, 1994). Expression of a tumor suppressor gene can alsobe abolished by de novo DNA methylation of a normally unmethylated CpGisland (Issa, et al., Nature Genet., 7:536, 1994; Herman, et al., supra;Merlo, et al., Nature Med., 1:686, 1995; Herman, et al., Cancer Res.,56:722, 1996; Graff, et al., Cancer Res., 55:5195, 1995; Herman, et al.,Cancer Res., 55:4525, 1995).

Human cancer cells typically contain somatically altered nucleic acid,characterized by mutation, amplification, or deletion of critical genes.In addition, the nucleic acid from human cancer cells often displayssomatic changes in DNA methylation (Fearon, et al., Cell, 61:759, 1990;Jones, et al., Cancer Res., 46:461, 1986; Holliday, Science, 238:163,1987; De Bustros, et al., Proc. Natl. Acad. Sci., USA, 85:5693, 1988);Jones, et al., Adv. Cancer Res., 54:1, 1990; Baylin, et al., CancerCells, 3:383, 1991; Makos, et al., Proc. Natl. Acad. Sci., USA, 89:1929,1992; Ohtani-Fujita, et al., Onco-gene, 8:1063, 1993). However, theprecise role of abnormal DNA methylation in human tumorogenesis has notbeen established.

Aberrant methylation of normally unmethylated CpG islands has beendescribed as a frequent event in immortalized and transformed cells, andhas been associated with transcriptional inactivation of defined tumorsuppressor genes in human cancers. This molecular defect has also beendescribed in association with various cancers. CpG islands are shortsequences rich in the CpG dinucleotide and can be found in the 5′ regionof about half of all human genes. Methylation of cytosine within 5′ CGIsis associated with loss of gene expression and has been seen inphysiological conditions such as X chromosome inactivation and genomicimprinting. Aberrant methylation of CpG islands has been detected ingenetic diseases such as the fragile-X syndrome, in aging cells and inneoplasia. About half of the tumor suppressor genes which have beenshown to be mutated in the germline of patients with familial cancersyndromes have also been shown to be aberrantly methylated in someproportion of sporadic cancers, including Rb, VHL, p16, hMLH1, and BRCA1(reviewed in Baylin, et al., Adv. Cancer Res. 72:141-196 1998).Methylation of tumor suppressor genes in cancer is usually associatedwith (1) lack of gene transcription and (2) absence of coding regionmutation. Thus CpG island methylation can serve as an alternativemechanism of gene inactivation in cancer.

Breast cancer is by far the most common form of cancer in women, and isthe second leading cause of cancer death in humans. Despite many recentadvances in diagnosing and treating breast cancer, the prevalence ofthis disease has been steadily rising at a rate of about 1% per yearsince 1940. Today, the likelihood that a woman living in North Americawill develop breast cancer during her lifetime is one in eight.

Breast cancer is often discovered at a stage that is advanced enough toseverely limit therapeutic options and survival rates. Accordingly, moresensitive and reliable methods are needed to detect small (less than 2cm diameter), early stage, in situ carcinomas of the breast. In additionto the problem of early detection, there remain serious problems indistinguishing between malignant and benign breast disease, in stagingknown breast cancers, and in differentiating between different types ofbreast cancers (e.g., estrogen dependent versus non-estrogen dependenttumors). Recent efforts to develop improved methods for breast cancerdetection have focused on cancer markers such as proteins that areuniquely expressed (e.g., as a cell surface or secreted protein) bycancerous cells, or are expressed at measurably increased or decreasedlevels by cancerous cells compared to normal cells. Accordingly, the useof the methylation status of certain genes as a marker of cancer orcancerous conditions provides an additional weapon in early detectionand prognosis of breast cancers.

Identification of the earliest genetic changes in cells associated withbreast cancer is a major focus in molecular cancer research. Diagnosticapproaches based on identification of these changes in specific genesare likely to allow implementation of early detection strategies andnovel therapeutic approaches. Targeting these early changes might leadto more effective cancer treatment.

SUMMARY OF THE INVENTION

The present invention is based on the finding that several genes arenewly identified as being differentially methylated in breast cancers.This seminal discovery is useful for breast cancer screening,risk-assessment, prognosis, disease identification, disease staging andidentification of therapeutic targets. The identification of new genesthat are methylated in breast cancer allows accurate and effective earlydiagnostic assays, methylation profiling using multiple genes; andidentification of new targets for therapeutic intervention.

In a first embodiment, the invention provides method of diagnosing acellular proliferative disorder of breast tissue in a subject comprisingdetermining the state of methylation of one or more nucleic acidsisolated from the subject. The state of methylation of one or morenucleic acids compared with the state of methylation of one or morenucleic acids from a subject not having the cellular proliferativedisorder of breast tissue is indicative of a cell proliferative disorderin the subject. In one aspect of this embodiment, the state ofmethylation is hypermethylation. The invention provides a method ofdiagnosing a cellular proliferative disorder of breast tissue in asubject by detecting the state of methylation of one or more of thefollowing nucleic acids: Twist, cyclin D2, WT1, NES-1, HOXA5 andcombinations thereof. Also methylated are RARβ2, 14.3.3 sigma, estrogenreceptor, RASSFIA, HIN-1 and combinations thereof. In one aspect of theinvention, nucleic acids are methylated in regulatory regions.

Invention methods can be used to diagnose disorders of the breastincluding breast cancers. In one aspect of the invention, disorders ofthe breast include ductal carcinoma in situ, lobular carcinoma, colloidcarcinoma, tubular carcinoma, medullary carcinoma, metaplasticcarcinoma, intraductal carcinoma in situ, lobular carcinoma in situ andpapillary carcinoma in situ.

Another embodiment of the invention provides a method of determining apredisposition to a cellular proliferative disorder of breast tissue ina subject. The method includes determining the state of methylation ofone or more nucleic acids isolated from the subject, wherein the stateof methylation of one or more nucleic acids compared with the state ofmethylation of the nucleic acid from a subject not having apredisposition to the cellular proliferative disorder of breast tissueis indicative of a cell proliferative disorder of breast tissue in thesubject. The nucleic acids can be nucleic acids encoding Twist, cyclinD2, RARβ2, HOXA5, WT, 14.3.3 sigma, estrogen receptor, NES-1, RASSFIA,HIN-1 and combinations thereof.

Still another embodiment of the invention provides a method fordetecting a cellular proliferative disorder of breast tissue in asubject. The method includes contacting a specimen containing at leastone nucleic acid from the subject with an agent that provides adetermination of the methylation state of at least one nucleic acid. Themethod further includes identifying the methylation states of at leastone region of at least one nucleic acid, wherein the methylation stateof the nucleic acid is different from the methylation state of the sameregion of nucleic acid in a subject not having the cellularproliferative disorder of breast tissue.

Yet a further embodiment of the invention provides a kit useful for thedetection of a cellular proliferative disorder in a subject comprisingcarrier means compartmentalized to receive a sample therein; and one ormore containers comprising a first container containing a reagent thatmodifies unmethylated cytosine and a second container containing primersfor amplification of a CpG-containing nucleic acid. The primershybridize with target polynucleotide sequence having the sequence ofcertain nucleic acids described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the nucleotide sequence of the cyclin D2 promoter (SEQ IDNO:105). Regions highlighted indicate primer sequences. CG nucleotidepairs are shown capitalized and bolded. A highlighted box shows thelocation of an atg codon. FIG. 1B shows nucleotide sequences for forward(F) and reverse (R) primer external and internal pairs used to detectmethylated (M) and unmethylated (U) nucleic acids. The base pair (BP)length of the primer pair product is also indicated.

FIGS. 2A and 2B show the nucleotide sequence of the TWIST promoter (SEQID NO:106). Regions highlighted indicate primer sequences. CG nucleotidepairs are shown capitalized and bolded. A highlighted box shows thelocation of an atg codon. FIG. 2C shows nucleotide sequences for forward(F) and reverse (R) external and internal primer pairs used to detectmethylated (M) and unmethylated (U) nucleic acids. The base pair (BP)length of the primer pair product is also indicated.

FIG. 3A shows the nucleotide sequence of the Retinoic Acid Receptor Beta(RARβ) promoter (SEQ ID NO:91). Regions highlighted indicate primersequences. CG nucleotide pairs are shown capitalized and bolded. Ahighlighted box shows the location of an atg codon. FIG. 3B showsnucleotide sequences for forward (F) and reverse (R) external andinternal primer pairs used to detect methylated (M) and unmethylated (U)nucleic acids. The base pair (BP) length of the primer pair product isalso indicated.

FIG. 4A shows the nucleotide sequence of Homo sapiens serineprotease-like protease (NES-1) mRNA. FIG. 4B shows the nucleotidesequence of the NES-1 region (exon 3) analyzed. Regions highlightedindicate primer sequences. CG nucleotide pairs are shown capitalized andbolded. FIG. 4C shows nucleotide sequences for forward (F) and reverse(R) primer pairs used to detect methylated (M) and unmethylated (U)nucleic acids. The base pair (BP) length of the primer pair product isalso indicated.

FIG. 5A shows the nucleotide sequence of HOXA5 promoter (3′ to 5′). CGnucleotide pairs are shown capitalized and bolded. A highlighted boxshows the location of a cat codon. FIG. 5B shows the nucleotide sequenceof the complementary region (5′ to 3″) analyzed (nucleotides −97 to−303). Regions highlighted indicate primer sequences. CG nucleotidepairs are shown capitalized and bolded. Highlighted box shows an atgcodon. FIG. 5C shows nucleotide sequences for forward (F) and reverse(R) primer pairs used to detect methylated (M) and unmethylated (U)nucleic acids. The base pair (BP) length of the primer pair product isalso indicated. FIG. 5D shows forward and reverse (sense and antisense)primers used for sequencing and expression of HOXA5.

FIG. 6A to 6F show the nucleotide sequence of Homo sapiens 14.3.3 sigmaprotein promoter and gene, complete cds.

FIGS. 7A and 7B show the nucleotide sequence of Homo sapiens Wilms'tumor (WT1) gene promoter.

FIGS. 8A and 8B show the nucleotide sequence of Homo sapiens estrogenreceptor beta gene, promoter region and partial cds. CG nucleotide pairsare shown capitalized and bolded. FIG. 8C shows nucleotide sequences offorward (F) and reverse (R) primer pairs used to detect methylated (M)and unmethylated (UM) nucleic acids. The base pair (BP) length of theprimer pair product is also indicated.

FIG. 9A shows the nucleotide sequence of human HIN-1 cDNA Regionshighlighted indicate primer sequences. FIG. 9B shows nucleotidesequences of forward and reverse external and internal primer pairs usedto detect methylated and unmethylated nucleic acids. The base pair (bp)length of the primer pair product is also indicated.

FIG. 10A shows the nucleotide sequence of the RASSF1A promoter. CGnucleotide pairs are shown capitalized and bolded. Regions highlightedindicate primer sequences. FIG. 10B shows nucleotide sequences offorward (F) and reverse (R) external and internal primer pairs used todetect methylated (M) and unmethylated (UM) nucleic acids. The base pair(BP) length of the primer pair product is also indicated.

FIG. 11 is a schematic representation of the invention assay methodsutilizing the technique of multiplex methylation-specific PCR.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based upon the discovery that the hypermethylation ofcertain genes can serve as markers for cellular proliferative disordersof breast tissue. This is the first time that promoter hypermethylationof certain genes such as Twist, cyclin D2, RARβ2, WT1, NES-1 and HOXA5have been associated with breast cancer.

It has been determined that the methylation state of nucleic acids ofcertain genes, particularly regulatory sequences, is diagnostic for thepresence or potential development of a cellular proliferative disorderof breast tissue in subjects. More particularly, the hypermethylation ofcertain nucleotides localized in CpG islands has been shown to affectthe expression of genes associated with the CpG islands; typically suchhypermethylated genes have reduced or abolished expression, primarilydue to down-regulated transcription. Hypermethylation of, for example,Twist, cyclin D2, retinoic acid receptor β (RARβ), WT1, HOXA5, 14.3.3sigma, estrogen receptor (ER) NES-1, the Ras association domain family1A gene (RASSF1A), and the high in normal-1 gene (HIN-1) allows one todiagnose a cellular proliferative disorder of breast tissue. Using arecently developed PCR-based technique called methylated specific PCR(MSP), aberrantly methylated nucleic acids in breast cancer primarytumors and biological samples from individuals with breast cancer can beidentified.

In a first embodiment, the invention provides a method of diagnosing acellular proliferative disorder of breast tissue in a subject comprisingdetermining the state of methylation of one or more nucleic acidsisolated from the subject, wherein the state of methylation of one ormore nucleic acids as compared with the state of methylation of one ormore nucleic acids from a subject not having the cellular proliferativedisorder of breast tissue is indicative of a cellular proliferativedisorder of breast tissue in the subject. A preferred nucleic acid is aCpG-containing nucleic acid, such as a CpG island.

A cell proliferative disorder as described herein may be a neoplasm.Such neoplasms are either benign or malignant. The term “neoplasm”refers to a new, abnormal growth of cells or a growth of abnormal cellsthat reproduce faster than normal. A neoplasm creates an unstructuredmass (a tumor), which can be either benign or malignant. The term“benign” refers to a tumor that is noncancerous, e.g. its cells do notinvade surrounding tissues or metastasize to distant sites. The term“malignant” refers to a tumor that is metastastic, invades contiguoustissue or no longer under normal cellular growth control.

One type of cellular proliferative disorder is a cell proliferativedisorder of breast tissue. Disorders of breast tissue or breast cancerscan involve numerous cells and tissues resulting in various disorders ofthe breast including ductal carcinoma in situ, lobular carcinoma,colloid carcinoma, tubular carcinoma, medullary carcinoma, metaplasticcarcinoma, intraductal carcinoma in situ, lobular carcinoma in situ, andpapillary carcinoma in situ.

The invention method includes determining the state of methylation ofone or more nucleic acids isolated from the subject. The phrases“nucleic acid” or “nucleic acid sequence” as used herein refer to anoligonucleotide, nucleotide, polynucleotide, or to a fragment of any ofthese, to DNA or RNA of genomic or synthetic origin which may besingle-stranded or double-stranded and may represent a sense orantisense strand, peptide nucleic acid (PNA), or to any DNA-like orRNA-like material, natural or synthetic in origin. As will be understoodby those of skill in the art, when the nucleic acid is RNA, thedeoxynucleotides A, G, C, and T are replaced by ribonucleotides A, G, C,and U, respectively.

The nucleic acid of interest can be any nucleic acid where it isdesirable to detect the presence of a differentially methylated CpGisland. The CpG island is a CpG rich region of a nucleic acid sequence.The nucleic acids includes, for example, a sequence encoding thefollowing genes (GenBank Accession Numbers are shown, followed by thenucleotides corresponding to the region(s) examined for the presence orabsence of methylation (numbers are relative to the first ATG codonunless otherwise indicated)): Twist (Accession No. AC003986; −51145 to151750 (complement) (SEQ ID NO:106), cyclin D2 (Accession No. U47284;−1616 to −1394) (SEQ ID NO:105); RARβ2 (Accession No. AF; 157484; −196to −357)(SEQ ID NO:91), WT1 (Accession No. AB034940) (SEQ ID NO: 103);HOXA5 (Accession No. AC004080) (SEQ ID NO:96), 14.3.3 sigma (AccessionNo. AF029081) (SEQ ID NO: 102); estrogen receptor (ER; Accession No.X62462) (SEQ ID NO:104); NES-1 (Accession No. AF024605) (SEQ ID NO:94);RASSF1A (Accession No. AF102770) (SEQ ID NO: 121); and HIN-1 (AccessionNo. AY040564) (SEQ ID NO: 120), the nucleotide sequence of each of whichis incorporated by reference herein.

WT1 encodes a transcriptional regulatory protein that binds DNA via fourCys₂His₂ zinc fingers. WT1 mRNA undergoes two independent splicingevents leading to the expression of at least four predominant isoforms.These splices result in the inclusion or omission of exon 5 (51 basepairs) and the presence or absence of a nine base pair insert (encodingthree amino acids, KTS) between the third and fourth zinc fingerdomains. Lack of expression has been observed in some Wilms” tumors,leading to classification as a tumor suppressor gene. However, WT1 isoverexpressed in 75% of cases of acute leukemia and is upregulated aschronic myeloid leukemia progresses into blast crisis. Thus, WT1 canapparently be either a tumor suppressor or an oncogene.

The cyclin D1, D2 and D3 proteins are involved in regulation of the cellcycle through phosphorylation and inactivation of the retinoblastomaprotein and activation of cyclin E, leading to transition of the cellsfrom G1 to DNA synthesis. In addition to their role in cell cycleregulation, the D-type cyclins have been implicated in differentiationand neoplastic transformation. Overexpression of cyclin D2 has beenreported in gastric cancer, and was shown to correlate with diseaseprogression and poor prognosis. Overexpression of cyclin D2 is alsonoted in ovarian granulosa cell tumors and testicular germ cell tumorcell lines.

14.3.3 σ is a member of a superfamily that is responsible forinstituting the G2 cell cycle checkpoint in response to DNA damage inhuman cells (Hermeking, et al. (1997) Mol. Cell 1, 3-11; Chan, et al.(1999) Nature 401, 616-20). In addition to any growth advantageresulting from a loosening of this checkpoint control mechanism, loss ofσ function is predicted to cause an increase in DNA damage in responseto γ-irradiation. Loss of 14.3.3. σ in primary epithelial cells leads toimmortalization (Dellambra et al. (2000) J. Cell Biol., 149:1117-1130),one of the earliest steps towards cancer.

Retinoic acid (RA) controls fundamental developmental processes, inducesterminal differentiation of myeloid progenitors and suppresses cancerand cell growth. RA activity is mediated by nuclear receptors, theretinoic acid receptors, RARs, that act as RA-dependent transcriptionalactivators in their heterodimeric forms with retinoid X receptors, RXRs(Chambon, 1996). RARs induce local chromatin changes at level of targetgenes, containing responsive RA elements (RAREs) by recruitingmultiprotein complexes with histone acetyltransferase (HAT) activity andhistone deacetylase (HDAC) activity that dynamically pattern chromatinmodification and regulate gene expression. RARs and RXRs, whendisrupted, result in severe developmental defects and neoplastictransformation. In breast cancer cells, the expression of one member ofthe RARs family, RAR β is found consistently down regulated or lost. RARβ downregulation can be reversed by RA in estrogen receptor(ER)-positive, but not in ER-negative breast carcinoma cell lines,believed to represent more advanced forms of tumors. Loss of RA-inducedRAR β expression is considered a crucial step in the development ofRA-resistance in breast carcinogenesis. A complex regulatory region,with two promoters, regulates RAR β gene expression. Only one promoter,RAR β2, containing several RA-response elements, including a canonicaland an auxiliary RA response element, βRARE, is active in human mammaryepithelial cells (HMEC). The transcription of the RAR β2 promoter ismediated by multiple RARs including, RARα and RAR β itself able torecruit coactivator and corepressor protein complexes with HAT/HDACactivities, respectively.

The Twist gene product is a transcription factor with DNA binding andhelix-loop-helix domains. Twist is a member of the bHLH transcriptionfactor family and is involved in the development of mesodermally derivedtissue including the skeleton. In humans, mutations in the Twist genehave been identified in patients with Saethre-chotzen syndrome, arelatively common craniosynostosis disorder with autosomal dominantinheritance. (see Gripp et al., (2000) Hum. Mutat. 15:479.) Twist alsoinfluences osteogenic gene expression and may act as a master switch ininitiating bone cell differentiation by regulating the osteogenic celllineage (Lee et al., (1999) J. Cell Biochem. 75:566-577).

NESI (normal epithelial cell-specific 1) is a novel gene with apredicted polypeptide of about 30.14 kilodaltons and having a 50-63%similarity and 34-42% identity with several families of serineproteases, in particular the trypsin-like proteases, members of theglandular kallikrein family (including prostate-specific antigen, nervegrowth factor gamma, and epidermal growth factor-binding protein) andthe activators for the kringle family proteins (including the humantissue plasminogen activator and human hepatocyte growth factoractivator) (Liu et al., (1996) Cancer Res. 56:14 3371-9). All of theresidues known to be crucial for substrate binding, specificity, andcatalysis by the serine proteases are conserved in the predicted NES1protein, indicating that it has protease-like activity.Immunolocalization studies with an antipeptide antibody directed againsta unique region of the NES1 protein (amino acids 120-137) detect aspecific 30-kilodalton polypeptide almost exclusively in the supernatantof the mRNA-positive mammary epithelial cells (MECs), suggesting thatNES1 is a secreted protein. The 1.4-kb NES1 mRNA is expressed in severalorgans (thymus, prostate, testis, ovary, small intestine, colon, heart,lung, and pancreas) with highest levels in the ovaries. Althoughexpression of the NES1 mRNA is observed in all normal and immortalizednontumorigenic MECs, the majority of human breast cancer cell lines showa drastic reduction or a complete lack of its expression. The structuralsimilarity of NES1 to polypeptides known to regulate growth factoractivity and a negative correlation of NES1 expression with breastoncogenesis suggest a direct or indirect role for this novelprotease-like gene product in the suppression of tumorogenesis. Studiesusing fluorescence in situ hybridization localized the NES1 gene tochromosome 19q13.3, a region that contains genes for related proteases(Goyal et al., (1998) Cancer Res., 58:21 4782-6).

The HOX genes are expressed during embryonic development and have a rolein specifying antero-posterior positional information. The genes arearranged in four clusters and a collinear relation exists between agene's position in the cluster and its anterior boundary of expression.Genes with more anterior boundaries are also expressed earlier thangenes with more posterior boundaries. Hox genes encode transcriptionfactors; therefore, a model for the coordinate regulation of the geneswithin the HOX clusters is that Hox gene products regulate their ownexpression. The production of HOXA5 from an expression vector canactivate a transient and simultaneous expression of other upstream anddownstream genes of the same HOX cluster and genes from other clusters.

The estrogen receptor gene has been implicated in the initiation and/orprogression of human breast cancer. Loss of expression of either genehas been associated with poorly differentiated tumors and poorerprognosis. Several studies have reported an association between estrogenreceptor (ER) expression and breast tumors. A loss of ER expression hasbeen associated with aberrant 5′ CpG island methylation in breast cancercell lines and primary human breast tumors. Studies show that aberrantmethylation of ER CpG islands begins before invasion of tumors intosurrounding tissues and it increases with metastatic progression (Naaset al., (2000) Cancer Res., 60:4346-4348; incorporated by reference inits entirety).

Hypermethylation of the CpG island of Ras Association Domain Family 1A(RASSF1A), a putative tumor suppressor gene from the 3p21.3 locus,occurs in a large percentage of human breast cancers. Hypermethylationof the RASSF1A promoter appears to be the main mechanism ofinactivation. The high frequency of epigenetic inactivation of theRASF1A gene in breast cancer supports its role as a putative tumorsuppressor gene (R. Dammann, et al., Cancer Research 61:3105-3109, 2001;K Dreijerink et al., PNAS 98(18):7504-7509, 2001; D. G. Burbee et al.,J. National Cancer Institute 93(9):691-699, 2001, each of which isincorporated herein by reference in its entirety).

Expression of HIN-1 (high in normal-1) is significantly down regulatedin 94% of human breast carcinoma and in 95% of preinvasive lesions, suchas ductal and lobular carcinoma in situ. This decrease in HIN-1expression is accompanied by hypermethylation of its promoter in themajority of breast cancer cell lines and primary tumors. This decreasein HIN-1 expression is accompanied by hypermethylation of its promoterin the majority of breast cancer cell lines (greater than 90%) andprimary tumors (74%). HIN-1 is a putative cytokine with no significanthomology to known proteins. Reintroduction of HIN-1 into breast cancelcells has been shown to inhibit cell growth, making HIN-1 a candidatetumor suppressor gene that is inactivated at high frequency in theearliest stages of breast tumorogenesis (I. E. Krop et al., PNAS98(17):9796-9801, 2001, which is incorporated herein by reference in itsentirety).

Any nucleic acid sample, in purified or nonpurified form, can beutilized in accordance with the present invention, provided it contains,or is suspected of containing, a nucleic acid sequence containing atarget locus (e.g., CpG-containing nucleic acid). One nucleic acidregion capable of being differentially methylated is a CpG island, asequence of nucleic acid with an increased density relative to othernucleic acid regions of the dinucleotide CpG. The CpG doublet occurs invertebrate DNA at only about 20% of the frequency that would be expectedfrom the proportion of G·C base pairs. In certain regions, the densityof CpG doublets reaches the predicted value; it is increased by ten foldrelative to the rest of the genome. CpG islands have an average G·Ccontent of about 60%, compared with the 40% average in bulk DNA. Theislands take the form of stretches of DNA typically about one to twokilobases long. There are about 45,000 such islands in the human genome.

In many genes, the CpG islands begin just upstream of a promoter andextend downstream into the transcribed region. Methylation of a CpGisland at a promoter usually prevents expression of the gene. Theislands can also surround the 5′ region of the coding region of the geneas well as the 3′ region of the coding region. Thus, CpG islands can befound in multiple regions of a nucleic acid sequence including upstreamof coding sequences in a regulatory region including a promoter region,in the coding regions (e.g., exons), downstream of coding regions in,for example, enhancer regions, and in introns.

In general, the CpG-containing nucleic acid is DNA. However, inventionmethods may employ, for example, samples that contain DNA, or DNA andRNA, including messenger RNA, wherein DNA or RNA may be single strandedor double stranded, or a DNA-RNA hybrid may be included in the sample. Amixture of nucleic acids may also be employed. The specific nucleic acidsequence to be detected may be a fraction of a larger molecule or can bepresent initially as a discrete molecule, so that the specific sequenceconstitutes the entire nucleic acid. It is not necessary that thesequence to be studied be present initially in a pure form; the nucleicacid may be a minor fraction of a complex mixture, such as contained inwhole human DNA. The nucleic acid-containing sample used fordetermination of the state of methylation of nucleic acids contained inthe sample or detection of methylated CpG islands may be extracted by avariety of techniques such as that described by Sambrook, et al.(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989;incorporated in its entirety herein by reference).

A nucleic acid can contain a regulatory region which is a region of DNAthat encodes information that directs or controls transcription of thenucleic acid. Regulatory regions include at least one promoter. A“promoter” is a minimal sequence sufficient to direct transcription, torender promoter-dependent gene expression controllable for cell-typespecific, tissue-specific, or inducible by external signals or agents.Promoters may be located in the 5′ or 3′ regions of the gene. Promoterregions, in whole or in part, of a number of nucleic acids can beexamined for sites of CG-island methylation.

Nucleic acids isolated from a subject are obtained in a biologicalspecimen from the subject. The nucleic acid may be isolated from breasttissue, blood, plasma serum, lymph, duct cells, nipple aspiration fluid,ductal lavage fluid and bone marrow. Tissue, blood, lymph, lymph node,duct cells, nipple aspiration fluid, ductal lavage fluid and bone marroware obtained by various medical procedures known to those of skill inthe art. Duct cells can be obtained by nipple aspiration, ducal lavage,sentinel node biopsy, fine needle aspirate, routine operative breastendoscopy and core biopsy. Ductal lavage fluid can be obtained by usinga DucWash procedure. In this procedure, a catheter is inserted into oneor more of the four to eight ducts typically present in each humanbreast, lavage of the duct is performed, and the lavage fluid iscollected. Alternatively, ductal lavage may be achieved through amicrocatheter procedure known as ROBE (routine operative breastendoscopy), which allows visualization of a tumor at the same time asaspiration of fluid from the duct.

In one aspect of the invention, the state of methylation in nucleicacids of the sample obtained from a subject is hypermethylation comparedwith the same regions of the nucleic acid in a subject not having thecellular proliferative disorder of breast tissue. Hypermethylation, asused herein, is the presence of methylated alleles in one or morenucleic acids. Nucleic acids from a subject not having a cellularproliferative disorder of breast tissues contain no detectablemethylated alleles when the same nucleic acids are examined.

A method for determining the methylation state of nucleic acids isdescribed in U.S. Pat. No. 6,017,704 which is incorporated herein in itsentirety and described briefly herein. Determining the methylation stateof the nucleic acid includes amplifying the nucleic acid by means ofoligonucleotide primers that distinguishes between methylated andunmethylated nucleic acids.

Two or more markers can also be screened simultaneously in a singleamplification reaction to generate a low cost, reliable cancer-screeningtest for breast cancers. A combination of DNA markers for CpG-richregions of nucleic acid may be amplified in a single amplificationreaction. The markers are multiplexed in a single amplificationreaction, for example, by combining primers for more than one locus. Forexample, DNA from a ductal lavage sample can be amplified with two ormore different unlabeled or randomly labeled primer sets in the sameamplification reaction. Especially useful are two or more markersselected from cyclin D2, RARβ2, Twist, NES-1, RASSF1A and HIN-1. Thereaction products are separated on a denaturing polyacrylamide gel, forexample, and then exposed to film or stained with ethidium bromide forvisualization and analysis. By analyzing a panel of markers, there is agreater probability of producing a more useful methylation profile for asubject.

For example, a screening technique, referred to herein as “multiplexmethylation-specific PCR” is a unique version of methylation-specificPCR. Methylation-specific PCR is described in U.S. Pat. Nos.5,786,146,6,200,756, 6,017,704 and 6,265,171, each of which is incorporated hereinby reference in its entirety. Multiplex methylation-specific PCRutilizes MSP primers for a multiplicity of markers, for example up tofive different breast cancer markers, in a two-stage nested PCRamplification reaction. The primers used in the first PCR reaction areselected to amplify a larger portion of the target sequence than theprimers of the second PCR reaction. The primers used in the first PCRreaction are referred to herein as “external primers” or DNA primers”and the primers used in the second PCR reaction are referred to hereinas “MSP primers.” Two sets of primers (i.e., methylated and unmethylatedfor each of the markers targeted in the reaction) are used as the MSPprimers. In addition in multiplex methylation-specific PCR, as describedherein, a small amount (i.e., 1 μl) of a 1:10¹ to about 10⁶ dilution ofthe reaction product of the first “external” PCR reaction is used in thesecond “internal” MSP PCR reaction. The technique of multiplexmethylation-specific PCR is illustrated schematically in FIG. 11.

As shown in Table 1 below, multiplex methylation-specific PCR greatlyenhances the accuracy of diagnosis obtainable from an amount of DNAavailable for analysis as compared with direct PCR analysis. TABLE 1 DNAMethod of PCR Sample Useage calculations Test Capacity DIRECT MSP: 20 μlDNA 1 μl per PCR rxn. 2 μl per test. If all 20 μl DNA (≦1 μg) Sufficientfor 20 rxns (10 sample is used, 10 tests); tests evaluate 5 genes × 2 2replicate tests of 5 genes. MULTIPLEX 20 μl DNA 2 μl per 1^(st) PCR rxn(25 μl If 2 μl DNA MSP: (≦1 μg) PCR rxn). sample is used, 125 1 μl 10¹dilution into 2^(nd) PCR tests evaluate 5 genes × 25. rxn (≦1 μg) If all20 μl starting DNA is used in multiplex methylation-specific PCR, up to10 panels of 5 genes × 25 replicates. 2 μl starting DNA is sufficientfor 250 2^(nd) PCR rxns (0.1 μl/rxn, 2 rxn/test, 125 tests from 25 μl1^(st) rxn

Multiplex methylation-specific PCR is also high specific. Testsconducted to compare the results of direct MSP with multiplexmethylation-specific PCR in analysis of the methylation status of humanprimary breast tumor, and human breast cancer cell lines, aresummarized, respectively, in Tables 5-7 below. The results shown inTables 5-7 illustrate concordance in the results obtained by analysis ofthese various types of samples using direct MPC and multiplexmethylation-specific PCR, as disclosed herein.

If the sample is impure (e.g., plasma, serum, lymph, ductal cells,nipple aspiration fluid, ductal lavage fluid, bone marrow, blood orbreast tissue embedded in paraffin), it may be treated beforeamplification with a reagent effective for lysing the cells contained inthe fluids, tissues, or animal cell membranes of the sample, and forexposing the nucleic acid(s) contained therein. Methods for purifying orpartially purifying nucleic acid from a sample are well known in the art(e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, 1989, herein incorporated by reference).

Primers hybridize with target polynucleotide sequences. Nucleic acidsequences including exemplary primers are set forth in SEQ ID NO: 1 toSEQ ID NO: 128. Oligonucleotide primers specifically targeted tomethylated and unmethylated genes including Twist, cyclinD2, RARβ2, WT1,HOXA5, 14.3.3 sigma, estrogen receptor, NES-1, RASSF1A, HIN-1, and theirassociated CpG islands include, respectively, SEQ ID NO:7-14, 21-24,37-40, 49-64, 69-72, 77-80, 85-90, 107-110, 116-119, 124-128, 129-130,and 135-136. (See Table 4 below).

Detection of differential methylation can be accomplished by contactinga nucleic acid sample with a methylation sensitive restrictionendonuclease that cleaves only unmethylated CpG sites under conditionsand for a time to allow cleavage of unmethylated nucleic acid. Thesample is further contacted with an isoschizomer of the methylationsensitive restriction endonuclease that cleaves both methylated andunmethylated CpG-sites under conditions and for a time to allow cleavageof methylated nucleic acid. Oligonucleotides are added to the nucleicacid sample under conditions and for a time to allow ligation of theoligonucleotides to nucleic acid cleaved by the restrictionendonuclease, and the digested nucleic acid is amplified by conventionalmethods, such as PCR wherein primers complementary to theoligonucleotides are employed. Following identification, the methylatedCpG-containing nucleic acid can be cloned, using methods well known tothose of skill in the art (see Sambrook et al., Molecular Cloning: ALaboratorv Manual, Cold Spring Harbor Press, 1989).

As used herein, a “methylation sensitive restriction endonuclease” is arestriction endonuclease that includes CG as part of its recognitionsite and has altered activity when the C is methylated as compared towhen the C is not methylated. Preferably, the methylation sensitiverestriction endonuclease has inhibited activity when the C is methylated(e.g., Smal). Specific non-limiting examples of methylation sensitiverestriction endonucleases include Sma I, BssHII, or HpaII, MspI, BSTUI,and NotI. Such enzymes can be used alone or in combination. Othermethylation sensitive restriction endonucleases will be known to thoseof skill in the art and include, but are not limited to SacII, and EagI,for example. An “isoschizomer” of a methylation sensitive restrictionendonuclease is a restriction endonuclease that recognizes the samerecognition site as a methylation sensitive restriction endonuclease butcleaves both methylated and unmethylated CGs. Those of skill in the artcan readily determine appropriate conditions for a restrictionendonuclease to cleave a nucleic acid (see Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Press, 1989).

A nucleic acid of interest is cleaved with a methylation sensitiveendonuclease. Cleavage with the methylation sensitive endonucleasecreates a sufficient overhang on the nucleic acid of interest, i.e.,sufficient to allow specific hybridization of an oligonucleotide ofinterest. Following cleavage with the isoschizomer, the cleavage productcan still have a sufficient overhang. An “overhang” refers to nucleicacid having two strands wherein the strands end in such a manner that afew bases of one strand are not base paired to the other strand. A“sufficient overhang” refers to an overhang of at least two bases inlength or four or more bases in length. An overhang of a specificsequence on the nucleic acid of interest may be desired in order for anoligonucleotide of interest to hybridize. In this case, the isoschizomercan be used to create the overhang having the desired sequence on thenucleic acid of interest.

Cleavage with a methylation sensitive endonuclease results in a reactionproduct of the nucleic acid of interest that has a blunt end or aninsufficient overhang. “Blunt end” refers to a flush ending of twostands, the sense stand and the antisense strand, of a nucleic acid.Once a sufficient overhang is created on the nucleic acid of interest,an oligonucleotide is ligated to the nucleic acid of interest, which hasbeen cleaved by the methylation specific restriction endonuclease.“Ligation” is the attachment of two nucleic acid sequences by basepairing of substantially complementary sequences and/or by the formationof covalent bonds between two nucleic acid sequences.

An adaptor can be utilized to create DNA ends of desired sequence andoverhang. An “adaptor” is a double-stranded nucleic acid sequence withone end that has a sufficient single-stranded overhang at one or bothends such that the adaptor can be ligated by base-pairing to asufficient overhang on a nucleic acid of interest that has been cleavedby a methylation sensitive restriction enzyme or an isoschizomer of amethylation sensitive restriction enzyme. Adaptors can be obtainedcommercially. Alternatively, two oligonucleotides that are substantiallycomplementary over their entire sequence except for the region(s) at the5′ and/or 3′ ends that will form a single stranded overhang can be usedto form an adaptor. The single stranded overhang on the adapter isselected to be complementary to an overhang on the nucleic acid cleavedby a methylation sensitive restriction enzyme or an isoschizomer of amethylation sensitive restriction enzyme, such that the overhang on thenucleic acid of interest will base pair with the 3′ or 5′ singlestranded end of the adaptor under appropriate conditions. The conditionswill vary depending on the sequence composition (GC vs AT), the length,and the type of nucleic acid (see Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press,Plainview, N.Y., 1998).

Following the ligation of the oligonucleotide to the nucleic acid ofinterest, the nucleic acid of interest is amplified using a primercomplementary to the oligonucleotide. Specifically, the term “primer” asused herein refers to a sequence comprising two or moredeoxyribo-nucleotides or ribonucleotides, preferably more than three,and more preferably more than eight, wherein the sequence is capable ofinitiating synthesis of a primer extension product that is substantiallycomplementary to a nucleic acid such as an adaptor or a ligatedoligonucleotide. Environmental conditions conducive to synthesis includethe presence of nucleoside triphosphates, an agent for polymerization,such as DNA polymerase, and suitable temperature and pH. The primer ispreferably single stranded for maximum efficiency in amplification, butmay be double stranded. If double stranded, the primer is first treatedto separate its strands before being used to prepare extension products.The primer can be an oligodeoxyribonucleotide. The primer must besufficiently long to prime the synthesis of extension products in thepresence of the agent for polymerization. The exact length of the primerwill depend on many factors, including temperature, buffer composition(i.e., salt concentration), and nucleotide composition. Theoligonucleotide primer typically contains 12-20 or more nucleotides,although it may contain fewer nucleotides.

Primers of the invention are designed to be “substantially”complementary to each strand of the oligonucleotide to be amplified andinclude the appropriate G or C nucleotides as discussed above. Thismeans that the primers must be sufficiently complementary to hybridizewith their respective strands under conditions that allow the agent forpolymerization to perform. In other words, the primers should havesufficient complementarity with a 5′ and 3′ oligonucleotide to hybridizetherewith and permit amplification of CpG containing nucleic acidsequence.

Primers of the invention are employed in the amplification process,which is an enzymatic chain reaction that produces exponentiallyincreasing quantities of target locus relative to the number of reactionsteps involved (e.g., polymerase chain reaction or PCR). Typically, oneprimer is complementary to the negative (−) strand of the locus(antisense primer) and the other is complementary to the positive (+)strand (sense primer). Annealing the primers to denatured nucleic acidfollowed by extension with an enzyme, such as the large fragment of DNAPolymerase I (Klenow) and nucleotides, results in newly synthesized +and − strands containing the target locus sequence. Because these newlysynthesized sequences are also templates, repeated cycles of denaturing,primer annealing, and extension results in exponential production of theregion (i.e., the target locus sequence) defined by the primer. Theproduct of the chain reaction is a discrete nucleic acid duplex withtermini corresponding to the ends of the specific primers employed.

The oligonucleotide primers used in invention methods may be preparedusing any suitable method, such as conventional phosphotriester andphosphodiester methods or automated embodiments thereof. In one suchautomated embodiment, diethylphos-phoramidites are used as startingmaterials and may be synthesized as described by Beaucage, et al.(Tetrahedron Letters, 22:1859-1862, 1981). One method for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066.

Another method for detecting a methylated CpG-containing nucleic acidincludes contacting a nucleic acid-containing specimen with an agentthat modifies unmethylated cytosine, amplifying the CpG-containingnucleic acid in the specimen by means of CpG-specific oligonucleotideprimers, wherein the oligonucleotide primers distinguish betweenmodified methylated and non-methylated nucleic acid and detecting themethylated nucleic acid. The amplification step is optional and althoughdesirable, is not essential. The method relies on the PCR reactionitself to distinguish between modified (e.g., chemically modified)methylated and unmethylated DNA.

The term “modifies” as used herein means the conversion of anunmethylated cytosine to another nucleotide that will facilitate methodsto distinguish the unmethylated from the methylated cytosine.Preferably, the agent modifies unmethylated cytosine to uracil.Preferably, the agent used for modifying unmethylated cytosine is sodiumbisulfite; however, other agents that similarly modify unmethylatedcytosine, but not methylated cytosine, can also be used in the method.Sodium bisulfite (NaHSO₃) reacts readily with the 5,6-double bond ofcytosine, but poorly with methylated cytosine. Cytosine reacts with thebisulfite ion to form a sulfonated cytosine reaction intermediate thatis susceptible to deamination, giving rise to a sulfonated uracil. Thesulfonate group can be removed under alkaline conditions, resulting inthe formation of uracil. Uracil is recognized as a thymine by Taqpolymerase. Therefore after PCR, the resultant product contains cytosineonly at the position where 5-methylcytosine occurs in the startingtemplate DNA.

The primers used in the invention for amplification of theCpG-containing nucleic acid in the specimen, after bisulfitemodification, specifically distinguish between untreated or unmodifiedDNA, methylated, and non-methylated DNA. MSP primers for thenon-methylated DNA preferably have a T in the 3′ CG pair to distinguishit from the C retained in methylated DNA, and the complement is designedfor the antisense primer. MSP primers usually contain relatively few Csor Gs in the sequence since the Cs will be absent in the sense primerand the Gs absent in the antisense primer (C becomes modified to U(uracil) which is amplified as T (thymidine) in the amplificationproduct).

The primers of the invention embrace oligonucleotides of sufficientlength and appropriate sequence so as to provide specific initiation ofpolymerization on a significant number of nucleic acids in thepolymorphic locus. Where the nucleic acid sequence of interest containstwo strands, it is necessary to separate the strands of the nucleic acidbefore it can be used as a template for the amplification process.Strand separation can be effected either as a separate step orsimultaneously with the synthesis of the primer extension products. Thisstrand separation can be accomplished using various suitable denaturingconditions, including physical, chemical, or enzymatic means, the word“denaturing” includes all such means. One physical method of separatingnucleic acid strands involves heating the nucleic acid until it isdenatured. Typical heat denaturation may involve temperatures rangingfrom about 80° to 105° C. for times ranging from about 1 to 10 minutes.Strand separation may also be induced by an enzyme from the class ofenzymes known as helicases or by the enzyme RecA, which has helicaseactivity, and in the presence of riboATP, is known to denature DNA. Thereaction conditions suitable for strand separation of nucleic acids withhelicases are described by Kuhn Hoffmann-Berling (CSH-QuantitativeBiology, 43:63, 1978) and techniques for using RecA are reviewed in C.Radding (Ann. Rev. Genetics, 16:405-437, 1982).

When complementary strands of nucleic acids are separated, regardless ofwhether the nucleic acid was originally double or single stranded, theseparated strands are ready to be used as a template for the synthesisof additional nucleic acid strands. This synthesis is performed underconditions allowing hybridization of primers to templates to occur.Generally synthesis occurs in a buffered aqueous solution, generally ata pH of about 7-9. Preferably, a molar excess (for genomic nucleic acid,usually about 108:1 primer:template) of the two oligonucleotide primersis added to the buffer containing the separated template strands. It isunderstood, however, that the amount of complementary strand may not beknown if the process of the invention is used for diagnosticapplications, so that the amount of primer relative to the amount ofcomplementary strand cannot be determined with certainty. As a practicalmatter, however, the amount of primer added will generally be in molarexcess over the amount of complementary strand (template) when thesequence to be amplified is contained in a mixture of complicatedlong-chain nucleic acid strands. Large molar excess is preferred toimprove the efficiency of the process.

The deoxyribonucleoside triphosphates dATP, dCTP, dGTP, and dTTP areadded to the synthesis mixture, either separately or together with theprimers, in adequate amounts and the resulting solution is heated toabout 90°-100° C. from about 1 to 10 minutes, preferably from 1 to 4minutes. After this heating period, the solution is allowed to cool toapproximately room temperature, which is preferable for the primerhybridization. To the cooled mixture is added an appropriate agent foreffecting the primer extension reaction (called herein “agent forpolymerization”), and the reaction is allowed to occur under conditionsknown in the art. The agent for polymerization may also be addedtogether with the other reagents if it is heat stable. This synthesis(or amplification) reaction may occur at room temperature up to atemperature above which the agent for polymerization no longerfunctions. Thus, for example, if DNA polymerase is used as the agent,the temperature is generally no greater than about 40° C. Mostconveniently the reaction occurs at room temperature.

The agent for polymerization may be any compound or system that willfunction to accomplish the synthesis of primer extension products,including enzymes. Suitable enzymes for this purpose include, forexample, E. coli DNA polymerase I, Klenow fragment of E. coli DNApolymerase I, T4 DNA polymerase, other available DNA polymerases,polymerase muteins, reverse transcriptase, and other enzymes, includingheat-stable enzymes (i.e., those enzymes which perform primer extensionafter being subjected to temperatures sufficiently elevated to causedenaturation such as Taq DNA polymerase, and the like). Suitable enzymeswill facilitate combination of the nucleotides in the proper manner toform the primer extension products that are complementary to each locusnucleic acid strand. Generally, the synthesis will be initiated at the3′ end of each primer and proceed in the 5′ direction along the templatestrand, until synthesis terminates, producing molecules of differentlengths. There may be agents for polymerization, however, which initiatesynthesis at the 5′ end and proceed in the other direction, using thesame process as described above.

Preferably, the method of amplifying is by PCR, as described herein andas is commonly used by those of ordinary skill in the art. However,alternative methods of amplification have been described and can also beemployed. PCR techniques and many variations of PCR are known. Basic PCRtechniques are described by Saiki et al. (1988 Science 239:487-491) andby U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, each of which isincorporated herein by reference.

The conditions generally required for PCR include temperature, salt,cation, pH and related conditions needed for efficient copying of themaster-cut fragment. PCR conditions include repeated cycles of heatdenaturation (i.e. heating to at least about 95° C.) and incubation at atemperature permitting primer: adaptor hybridization and copying of themaster-cut DNA fragment by the amplification enzyme. Heat stableamplification enzymes like the pwo, Thermus aquaticus or Thermococcuslitoralis DNA polymerases which eliminate the need to add enzyme aftereach denaturation cycle, are commercially available. The salt, cation,pH and related factors needed for enzymatic amplification activity areavailable from commercial manufacturers of amplification enzymes.

As provided herein an amplification enzyme is any enzyme which can beused for in vitro nucleic acid amplification, e.g. by theabove-described procedures. Such amplification enzymes include pwo,Escherichia coli DNA polymerase I, Klenow fragment of E. coli DNApolymerase I, T4 DNA polymerase, T7 DNA polymerase, Thermus aquaticus(Taq) DNA polymerase, Thermococcus litoralis DNA polymerase, SP6 RNApolymerase, T7 RNA polymerase, T3 RNA polymerase, T4 polynucleotidekinase, Avian Myeloblastosis Virus reverse transcriptase, Moloney MurineLeukemia Virus reverse transcriptase, T4 DNA ligase, E. coli DNA ligaseor Qβ replicase. Preferred amplification enzymes are the pwo and Taqpolymerases. The pwo enzyme is especially preferred because of itsfidelity in replicating DNA.

Once amplified, the nucleic acid can be attached to a solid support,such as a membrane, and can be hybridized with any probe of interest, todetect any nucleic acid sequence. Several membranes are known to one ofskill in the art for the adhesion of nucleic acid sequences. Specificnon-limiting examples of these membranes include nitrocellulose(NITROPURE®) or other membranes used in for detection of gene expressionsuch as polyvinylchloride, diazotized paper and other commerciallyavailable membranes such as GENESCREEN®, ZETAPROBE® (Biorad), andNYTRAN®. Methods for attaching nucleic acids to these membranes are wellknown to one of skill in the art. Alternatively, screening can be donein a liquid phase.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

An example of progressively higher stringency conditions is as follows:2×SSC/0.1% SDS at about room temperature (hybridization conditions);0.2×SSC/0.1% SDS at about room temperature (low stringency conditions);0.2×SSC/0.1% SDS at about 42° C. (moderate stringency conditions); and0.1×SSC at about 68° C. (high stringency conditions). Washing can becarried out using only one of these conditions, e.g., high stringencyconditions, or each of the conditions can be used, e.g., for 10-15minutes each, in the order listed above, repeating any or all of thesteps listed. However, as mentioned above, optimal conditions will vary,depending on the particular hybridization reaction involved, and can bedetermined empirically. In general, conditions of high stringency areused for the hybridization of the probe of interest.

The probe of interest can be detectably labeled, for example, with aradioisotope, a fluorescent compound, a bioluminescent compound, achemiluminescent compound, a met al chelator, or an enzyme. Those ofordinary skill in the art will know of other suitable labels for bindingto the probe, or will be able to ascertain such, using routineexperimentation.

Another embodiment of the invention provides a method of determining apredisposition to a cellular proliferative disorder of breast tissue ina subject comprising determining the state of methylation of one or morenucleic acids isolated from the subject, wherein the nucleic acid isselected from the group consisting of Twist, cyclin D2, RARβ2, HOXA5,WT1, 14.3.3 sigma, estrogen receptor, NES-1, RASSF1A, HIN-1, andcombinations thereof; and wherein the state of methylation of one ormore nucleic acids as compared with the state of methylation of saidnucleic acid from a subject not having a predisposition to the cellularproliferative disorder of breast tissue is indicative of a cellproliferative disorder of breast tissue in the subject.

As used herein, “predisposition” refers to an increased likely that anindividual will have a disorder. Although a subject with apredisposition does not yet have the disorder, there exists an increasedpropensity to the disease.

Another embodiment of the invention provides a method for diagnosing acellular proliferative disorder of breast tissue in a subject comprisingcontacting a nucleic acid-containing specimen from the subject with anagent that provides a determination of the methylation state of nucleicacids in the specimen, and identifying the methylation state of at leastone region of least one nucleic acid, wherein the methylation state ofat least one region of at least one nucleic acid that is different fromthe methylation state of the same region of the same nucleic acid in asubject not having the cellular proliferative disorder is indicative ofa cellular proliferative disorder of breast tissue in the subject.

Invention methods are ideally suited for the preparation of a kit.Therefore, in accordance with another embodiment of the presentinvention, there is provided a kit it useful for the detection of acellular proliferative disorder in a subject. Invention kits include acarrier means compartmentalized to receive a sample therein, one or morecontainers comprising a first container containing a reagent whichmodifies unmethylated cytosine and a second container containing primersfor amplification of a CpG-containing nucleic acid, wherein the primersdistinguish between modified methylated and nonmethylated nucleic acid.Primers contemplated for use in accordance with the invention includethose set forth in SEQ ID NOs: 7-14, 21-24, 37-40, 49-64, 69-72, 77-80,85-90, 116-119, 124-128, and combinations thereof.

Carrier means are suited for containing one or more container means suchas vials, tubes, and the like, each of the container means comprisingone of the separate elements to be used in the method. In view of thedescription provided herein of invention methods, those of skill in theart can readily determine the apportionment of the necessary reagentsamong the container means. For example, one of the container means cancomprise a container containing an oligonucleotide for ligation tonucleic acid cleaved by a methylation sensitive restrictionendonuclease. One or more container means can also be includedcomprising a primer complementary to the oligonucleotide. In addition,one or more container means can also be included which comprise amethylation sensitive restriction endonuclease. One or more containermeans can also be included containing an isoschizomer of saidmethylation sensitive restriction enzyme.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples, which are provided herein for purposes ofillustration only and are not intended to limit the scope of theinvention.

Although the invention has been described with reference to thepresently preferred embodiment, is should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

EXAMPLE 1 Methylation Status of Wilms' Tumor Suppressor Gene (WT1)

The extent of methylation of the WT1-associated CpG islands in normalmammary epithelium, in breast cancer cell lines, and in primary mammarytumors, and expression of the WT1 mRNA and protein in the same cells andtissues was examined.

Cell lines and finite life span cultures Cell lines were obtained fromATCC (Rockville, Md.) and grown according to conditions specified. Alsoutilized were three independent cultures of finite life span humanmammary epithelial cells (HMEC): 16637 (Clonetics, Walkersville, Md.)and 1-26, 3-14 (kindly provided by Dr. Steve Ethier, Univ. Michigan, AnnArbor, Mich.). When indicated, cell lines were treated with 0.75 μM5-aza-2′-deoxycytidine (5-aza-dC) or with 100 ng/ml Trichostatin A (TSA)as described in Ferguson, et al. (Proc Natl Acad Sci USA. (2000)97:6049-54).

Tumors and Organoids Primary breast tumors were obtained from the JohnsHopkins frozen tumor bank. Mammary organoids were prepared fromreduction mammoplasty specimens of women with benign (B) or no (N)abnormalities in the breast as described in Fujii, et al. (Oncogene. 16:2159-64, 1998). Briefly, the specimens were enzymatically digested intoduct-like structures (organoids), filtered, histologically confirmed tocontain more than 80% epithelial cells, and frozen at −70° C. untilused. Also utilized were highly purified myo- and luminal epithelialcells isolated by differential centrifugation and fluorescence-activatedcell sorting of enzymatically digested normal mammoplasty specimens(Gomm, et al., (1995) Anal Biochem. 226 91-9).

RT-PCR for WT1 mRNA Methods for RNA extraction and RT-PCR are known tothose of skill in the art. The sequences of the primers used are asfollows: for amplifying the 555 bp region surrounding WTI exon 5,5′-GCGGCGCAGTTCCCCAACCA-3′ (sense, nucleotides 882-901; SEQ ID NO: 1)and 5′-ATGGTTTCTCACCAGTGTGCTT-3′ (antisense, nucleotides 1416-1437; SEQID NO:2); for amplifying the 382 bp region surrounding the KTS insert,5′-GCATCTGAAACCAGTGAGAA-3′ (sense, nucleotides 1320-1339; SEQ ID NO:3)and 5′-TTTCTCTGATGCATGTTG-3′ (antisense, nucleotides 1685-1702; SEQ IDNO:4). Amplification was performed using a hot-start protocol: sampleswere heated to 94° C. for 4 minutes and then cooled to 80° C. prior tothe addition of Taq polymerase (RedTaq, Sigma, St. Louis, Mo.). Sampleswere then heated to 94° C. for 30 seconds followed by either 50° C. for30 seconds (for the KTS primers) or 56° C. for 30 seconds (for the Exon5 primers) and then 72° C. for 1 minute for 40 cycles. PCR products wereresolved by electrophoresis, using a 2% agarose gel for the exon5-splice variants and a 12% polyacrylamide gel to resolve the KTS insertvariants. Co-amplification of the ribosomal RNA 36B4 was performed as aninternal control using the following primers:5′-GATTGGCTACCCAACTGTTGCA-3′ (sense; SEQ ID NO:5) and5′-CAGGGGCAGCAGCCACAAAGGC-3′. (antisense; SEQ ID NO:6)

Northern blots Total RNA was extracted as described above. Afterelectrophoresis through a 1.5% agarose gel in MOPS buffer with 6.7%formaldehyde, RNA was transferred to nitrocellulose. Blots were probedwith a PCR product corresponding to the WT1 zinc finger region,amplified using the primers described above and labeled by randompriming using standard techniques.

Methylation-specific PCR Genomic DNA was isolated using standardtechniques and treated with sodium bisulfite as described elsewhere(Herman, et al., Proc Natl Acad Sci USA. 93: 9821-6, 1996).Methylation-specific PCR was performed using the following primers: todetect methylated promoter DNA, 5′-TTTGGGTTAAGTTAGGCGTCGTCG-3′ (sense;SEQ ID NO:7) and 5′-ACACTACTCCTCGTACGACTCCG-3′ (antisense; SEQ ID NO:8);to detect unmethylated promoter DNA, 5′-TTTGGGTTAAGTTAGGTGTTGTTG-3′(sense; SEQ ID NO:9) and 5′-ACACTACTCCTCATACAACTCCA-3′ (antisense; SEQID NO:10); to detect methylated intron 1 DNA,5′-CGTCGGGTGAAGGCGGGTAAT-3′ (sense; SEQ ID NO:11) and5′-CGAACCCGAACCTACGAAACC-3′ (antisense; SEQ ID NO:12); to detectunmethylated intron 1 DNA, 5′-TGTTGGGTGAAGGTGGGTAAT-3′ (sense; SEQ IDNO: 13) and 5′-CAAACCCAAACCTACAAAACC-3′ (antisense; SEQ ID NO: 14). ThePCR reaction was as described above, except that the annealingtemperature was 59° C., and the extension time was 45 seconds.

Western blots Total protein from cell lines was obtained from materialharvested in TriReagent (Molecular Research Center, Cincinnati, Ohio)and initially used for RNA isolation. Protein purification was accordingto the manufacturer's protocol. After separation by SDS-PAGE andelectrophoretic transfer to nitrocellulose membranes, proteins wereincubated with an anti-WT1 antibody [WT (C-19); sc-192, Santa CruzBiotechnology, Santa Cruz, Calif.] diluted 1:1000 in the blockingsolution. Horseradish peroxidase-conjugated antibody against rabbit IgG(Amersham, Arlington Heights, Ill.) was used at 1:1000, and binding wasrevealed using enhanced chemiluminescence (Amersharn, Arlington Heights,Ill.).

Expression of WT1 mRNA in mammary epithelial and breast cancer celllines To evaluate WT1 expression in the breast, mRNA expression wasanalyzed by RT-PCR in a panel of normal and transformed cell lines. NoWT1 RNA was detected in 3 independently derived finite lifespan mammaryepithelial strains: HMEC 16637, 1-26, and 3-14. Among the three immortalbreast epithelial cell lines, WT1 expression was observed in HMECsHBL-100 and MCF-10A, but not in H16N. WT1 mRNA expression was examinedin nine breast cancer cell lines, expression was easily detectable infive: HS578T, T47D, MDA-MB-468, 2IMT, and 21PT, and undetectable in theremaining four: SKBR3, MDA-MB-435, MCF-7 and MDA-MB-231.

The specific expression of WT1 isoforms lacking the fifth exon andlacking the KTS insert has been reported to occur in breast cancer(Silberstein, et al., Proc Natl Acad Sci USA. 94: 8132-7, 1997). Todetermine if differential expression of WT1 splice variants is seen inbreast cancer cell lines, PCR primers were designed spanning the fifthexon such that mRNA encoding the isoform containing exon 5 yielded a 555bp PCR product, while if exon 5 were missing a 504 bp PCR product wasgenerated. PCR primers spanning the region of the KTS insert, such thatan mRNA containing the insert would yield a 382 bp product, while an RNAlacking the insert would generate a 373 bp product were also used.Contrary to the findings in the published report (Silberstein et al.,supra), in the five WT1-expressing breast cancer cell lines, and in theWT1-expressing immortalized HMECs, all four splice variants-the two exon5 isoforms and the two KTS isoforms, were present.

To confirm these results, Northern blot analysis was performed usingtotal RNA isolated from a number of breast cancer cell lines. Similar tothe results obtained by RT-PCR, WT1 MnRNA expression was readilydetected in HBL-100, HS-578T, T47D, and MDA-MB-468 cells but was notdetected in MDA-MB-435, MDA-MB-231, SKBR3, or MCF-7 cells.

Thus, WT1 mRNA expression was undetectable in finite life span primarybreast epithelial cell cultures, but was easily detectable in theneoplastic and immortalized HMECs and in seven of twelve breast cancercell lines. Also, the striking correlation between results from Northernblots and RT-PCR experiments validated the RT-PCR protocol for thedetection of WT1 mRNA expression.

Methylation of the WT1 locus in breast cancer cell lines The promoterand first intron of the WT1 gene contain dense CpG islands. Thesesequence elements are frequently sites of DNA methylation, and play arole in transcriptional silencing (Nan, et al., Cell. 88: 471-81, 1997;Ng, et al., Nat Genet. 23: 58-61, 1999). To determine whethermethylation silences gene expression in the WT1-negative cell lines, thestatus of the WT1 promoter in the breast cancer cell lines wasinvestigated. The promoter was methylated in the 4 cell lines that didnot express WT1, but not in the 5 cell lines that did, consistent withthe idea that methylation is a critical determinant of WT1 expression.There was one exception to this correlation. T47D cells containedmethylated WT1 sequences but nevertheless expressed WT1 mRNA, suggestingthat, in this case, methylation alone is insufficient to silenceexpression.

Promoter methylation is postulated to silence transcription, at least inpart, by recruitment of histone deacetylase (HDAC) to hypermethylatedloci (Nan, et al. supra and Ng, et al., supra). In order to assess thefunctional significance of WT1 promoter methylation, MDA-MB-231 andMCF-7 cells were treated with 5-aza-deoxyC, an inhibitor of DNAmethyltransferases, or with TSA, an inhibitor of HDAC. As demonstratedbefore (Laux, et al., Breast Cancer Res Treat. 56: 35-43, 1999),treatment with 5-aza-deoxyC resulted in WT1 expression by MDA-MB-231cells. Interestingly, this treatment did not cause WT1 expression inMCF-7 cells, nor did TSA restore expression in either cell line. In thesame samples, these treatments restored expression of 14.3.3σ (Ferguson,et al., Proc Natl Acad Sci USA. (2000) 97:6049-54). These findingssuggest that while promoter methylation correlates with gene silencing,it may not play a causal role.

Expression of WT1 in primary breast tissue These findings from celllines were expanded to patient samples, including normal breastepithelium and primary breast tumors. Breast carcinomas arise fromluminal epithelial cells in the mammary duct. Normal breast tissue alsocontains a layer of myoepithelial cells, which overlie the luminalepithelium. To ensure that the normal samples contained luminalepithelial cells, three different types of epithelial cell preparationswere used, including (1) three short term cultures of HMECs, (2) nineorganoid preparations of mammary ducts, and (3) eight samples of highlypurified luminal and myoepithelial cells (isolated from 4 patientsamples).

WT1 expression was not detected by RT-PCR in 3 HMEC samples, in eightout of nine breast organoid preparations, nor in any of eight purifiedepithelial cell preparations. By western blotting, WT1 protein was notdetected in three organoid samples nor in two HMECs. In contrast, WT1expression was easily detectable in 27 out of 31 (87%) primary breastcarcinomas.

The HMECs did not express WTI; however, RT-PCR using primers describedabove demonstrated the expression of Exon 5 (+) and Exon 5 (−) isoformsin five out of seven tumors, while the remaining two expressed only theExon 5 (+) isoform. KTS (+) and KTS (−) isoforms were detected in allnine tumors examined. Thus, a majority of the tumors expressed both Exon5 splice variants of WT1, and all of the tumors express both splicevariants involving the KTS insert. Interestingly, the sole breastorganoid sample that expressed WT1 expressed all four splice variants aswell.

Methylation of WT1-associated CpG islands in normal and malignant breasttissue Since methylation of the promoter-associated CpG islandcorrelated with a lack of WT1 expression in breast cancer cell lines,the methylation status of the promoter and first intron CpG islands wasstudied in this panel of breast organoids and carcinomas. Prior studiesdemonstrating tumor-specific methylation of the CpG islands associatedwith the WT1 gene have employed methylation-sensitive restrictionenzymes (Huang, et al., Cancer Res. 57: 1030-4, 1997; Laux, et al.,Breast Cancer Res Treat. 56: 35-43, 1999; and Huang, et al., Hum MolGenet. 8: 459-70, 1999). This technique is a reliable way to identifyindividual methylated sites, but it is unable to assess large-scalemethylation patterns. The density of methylation, rather thanmethylation of any specific CpG dinucleotide, is responsible for genesilencing (Herman, et al., Semin Cancer Biol. 9: 359-67, 1999).Therefore, methylation of the CpG islands was evaluated usingmethylation specific PCR (MSP). This method allows the direct evaluationof several methylation sites per PCR reaction, and choosing a variety ofsequences for PCR primers allows the rapid assessment of many CpGdinucleotides (Herman, et al., Proc Natl Acad Sci USA. 93: 9821-6,1996).

MSP was performed using DNA extracted from 19 primary tumors and ninebreast organoid preparations. The WT1 promoter CpG island wasunmethylated in DNA from all nine organoid samples. In contrast, six of19 tumors contained methylated DNA, and the remaining 13 were completelyunmethylated. This rate of promoter methylation (32%) is not dissimilarto the 25% incidence reported by Laux et al. (Breast Cancer Res Treat.56: 35-43, 1999). Thus, methylation of the WT1 promoter is atumor-specific phenomenon. Contrary to expectation, however, each of thesix tumors that contained methylated WT1 also expressed WT1 protein. WT1gene methylation, therefore, was not effective in silencing geneexpression. Next, the CpG island in the first intron of the WT1 gene, aregion where tumor-specific methylation has also been previouslyreported was examined. Methylation of WT1 was detected in all threebreast organoid preparations and in nine of ten tumor samples evaluated.Thus, the first intron of WT1 is methylated in both normal and malignantbreast tissue, and is unrelated to tumorogenesis.

Methylation of the CpG island associated with the WT1 promoter isassociated with a gene silencing in several breast cancer cell lines.While treatment of MDA-MB-231 cells with the methyltransferase inhibitor5-aza-deoxyC results in re-expression of the gene, this was not seen inMCF-7 cells. Additionally, treatment with the HDAC inhibitor TSA had noeffect on WT1 expression, suggesting that DNA methylation and histoneacetylation play only minor roles in the regulation of WT1 expression inmammary epithelium.

This study demonstrates tumor-specific methylation of the CpG islands ofWT1. Surprisingly, expression of WT1 mRNA and protein in the majority ofbreast cancer samples evaluated was also found, including in everysample that contained methylated DNA. these findings that breastcarcinomas express WT1 despite tumor-specific gene methylationemphasizes the importance of evaluating methylation and gene expressionconcurrently in the same tissue.

WT1 mRNA was readily detected in tumor samples using a single step PCRprotocol. While it is possible to detect WT1 expression in normalepithelium using a nested PCR, this would not alter the finding that thegene is overexpressed in tumors compared with normal tissue. The use ofRT-PCR may allow the detection of a relatively weakly expressed gene,but WT1 protein was readily detected by Western blotting in tumors.Since protein is the functional species, this finding suggests that WT1is abundant enough in tumors to play a functional role.

These data also reveal a discrepancy between gene regulation in tissueculture and in vivo. Methylation of the WT1 promoter is associated withgene silencing in breast cancer cell lines. In contrast, thepromoter-associated CpG island was methylated in 32% of the tumorsexamined; contrary to expectation, these tumors express WT1. These datahighlight the fact that there are multiple mechanisms for genesilencing, of which hypermethylation of a CpG island is only one. Moreimportantly, these findings emphasize the idea that cell lines do notnecessarily reflect the in vivo situation. They also serve to point outthat hypermethylation of a CpG island may be insufficient to silenceexpression, demonstrating the importance of assessing gene expression aswell as promoter methylation status when evaluating the role of aparticular gene in a particular tumor type.

In summary, these data demonstrate that WT1 is not expressed in normalbreast epithelium and is over-expressed in the majority of primarybreast tumors. Tumor-specific methylation of the CpG island occurs inbreast cancer, but appears to be inconsequential to gene expression.

EXAMPLE 2 Hypermethylation and Loss of Expression of Cyclin D2

The extent of methylation of the cyclin D2-associated CpG islands innormal mammary epithelium, in breast cancer cell lines, and in primarymammary tumors, and expression of the cyclin D2 mRNA and protein in thesame cells and tissues was examined.

Cell Lines and Tissues The breast cancer cell lines MDAMB435, MCF7,T47D, SKBR3, ZR75.1, MDAMB468, HS578T, MDAMB231 and the immortal humanmammary epithelial cell lines (HMEC) MCF10A and HBL100 were obtained andmaintained in culture according to instructions (ATCC, Rockville, Md.).The two matched tumor cell lines, 21PT, derived from a primary tumor and21MT, from the metastasis of the same patient, were propagated asdescribed elsewhere. The breast cancer cell line, MW, was obtained fromDr. Renato Dulbecco. HMEC-H16N (immortalized with HPV) was kindlyprovided by Dr. Vimla Band. Cultured finite life span human breastepithelial cell strains 04372, 219-6, and 166372 were obtained fromClonetics (Walkersville, Md.), and HMEC strains 1-26 and 3-14 werekindly provided by Dr. Steve Ethier. Finite life span HMEC 184, theimmortalized HMECs 184A1 (passage 15 and 99) and 184B5 were kindlyprovided by Dr. Martha Stampfer, and grown as described on the worldwideweb site 1bl.gov/LBL-Programs/mrgs/review.html. Cell extracts fromfinite lifespan HMECs 70N and 81N were kindly provided by Dr. KhandanKeyomarsi. Mammary organoids were prepared from reduction mammoplastyspecimens of women with benign or no abnormalities in the breastfollowing collagenase digestion as described in Bergstraessar LM,(1993). Human mammary luminal and myoepithelial cells were prepared byprogressive collagenase digestion of breast tissue, sedimentated toobtain organoids (ductal and lobulo-alveolar fragments), cultured shortterm, and finally highly enriched by using an immunomagnetic separationtechnique (Niranjan B, 1995).

Primary breast tumor tissues were obtained after surgical resection atthe Johns Hopkins University and Duke University, and stored frozen at−80° C. Samples containing greater than 50% tumor cells were selectedfollowing microscopic examination of representative tissue sections fromeach tumor. Microdissection of carcinoma and ductal carcinomas in situ(DCIS) lesions from eight micron cryosections was performed by using alaser capture microscope, or by manually scraping the cells with a 25Gneedle under 40× magnification. Genomic DNA was extracted by incubatingthe microdissected cells at 55° C.×12 h in 50 μl buffer containing 10 mMTris Cl (pH 8.0), 1 mM EDTA, 0.1% Tween 20, and 0.5 μg/μl proteinase K.The extract was heat inactivated at 95° C. for 5 min., and used directlyfor sodium bisulfite treatment.

RT-PCR RNA was treated with RNAse-free DNAse (Boehringer-Mannheim)(0.5-1 u/ul) for 30 min. at 37° C., followed by heat inactivation at 65°C. for 10 min. RT reactions contained 2 μg DNAse treated RNA, 0.25 μg/μlpdN6 random primers (Pharmacia), 1× first strand buffer (GibcoBRL), 1 mMdNTP (Pharmacia), and 200 U MMLV-RT (GibcoBRL), and were incubated for 1h at 37° C. followed by heat inactivation at 75° C. for 5 min. PCR wasperformed using the primers 5′-CATGGAGCTGCTGTGCCACG-3′ (sense; SEQ IDNO:15) and 5′-CCGACCTACCTCCAGCATCC-3′ (antisense; SEQ ID NO:16) forcyclin D2 and primers 5′-AGCCATGGAACACCAGCTC-3′ (sense; SEQ ID NO:17)and 5′-GCACCTCCAGCATCCAGGT-3′ (antisense; SEQ ID NO:18) for cyclin D1.Co-amplified products of 36B4, a “housekeeping” ribosomal protein gene,was used as an internal control, using primers5′-GATTGGCTACCCAACTGTTGCA-3′ (sense; SEQ ID NO:19) and5′-CAGGGGCAGCAGCCACAAAGGC-3′ antisense; SEQ ID NO:20). The 25 μlreactions contained 1× buffer (2× Reaction Mix, cat # 10928-026, BRL)and 100 nM of each primer. The PCR conditions were: 1 cycle of 94° C.for 1 min “hot start” then addition of 1 u of Taq polymerase (RedTaq), 1cycle of 94° C. for 2 min, 35 cycles of: 94° C. for 15 sec, 55° C. for30 sec, 72° C. for 45 sec, and finally 72° C. for 5 min. The PC wereresolved by electrophoresis on a 2% agarose gel in 1× TBE buffer.

Methylation-specific PCR (MSP) One μg genomic DNA or the 50 ul extractof microdissected cells was treated with sodium bisulfite as describedin Herman JG, (1996), and was analyzed by MSP using primer sets locatedwithin the CpG-rich island in the cyclin D2 promoter. Primers specificfor unmethylated DNA were 5′-GTTATGTTATGTTTGTTGTATG-3′ (sense; SEQ IDNO:21) and 5′-GTTATGTTATGTTTGTTGTATG-3′ (antisense; SEQ ID NO:22) andyielded a 223 base-pairs PCR product. Primers specific for methylatedDNA were 5′-TACGTGTTAGGGTCGATCG-3′ (sense; SEQ ID NO:23) and5′-CGAAATATCTACGCTAAACG-3′ (antisense; SEQ ID NO:24) and yielded a 276base-pair PCR product. The PCR conditions were as follows: 1 cycle of95° C. for 5 min; 35 cycles of 95° C. for 30 s, 55° C. for 30 s and 72°C. for 45 s; and 1 cycle of 72° C. for 5 min. The PCR products wereresolved by electrophoresis in a 2% agarose gel in 1× TBE buffer.

Treatment of Cells with 5′-aza-2′-deoxycytidine (5-aza-dC) andTrichostatin A (TSA) Cells were seeded at a density of 1×10⁶ cells per100-mm plate. 24 h later cells were treated with 0.75 μM 5-aza-dC(Sigma) or with 100 ng/ml of TSA (Sigma). Total cellular DNA and RNAwere isolated at 0, 3 and 5 days after addition of 5-aza-dC and at 0, 24and 48 hours after addition of TSA, as described above.

Western Blot Analysis Proteins were extracted from cell pellets and from8 micron sections of primary breast tumors in buffer containing 20 mMTris pH 7.5, 150 nM NaCl and PMSF, and sonicated. Twenty gg of proteinwere fractionated on 12.5% SDS-PAGE and transferred by electrophoresisto a nylon membrane. The blot was incubated with anti-cyclin D2 antibody(Ab-4, “cocktail” mouse monoclonal antibodies, Neomarkers, San Diego,Calif.) diluted 1:200 in 5% skim milk, for 2 h at room temperature.Horseradish peroxidase-conjugated antibody anti-mouse IgG (Amersham) wasused at 1:1000, and binding was revealed using enhancedchemiluminescence (Amersham).

Cyclin D2 mRNA expression in breast cancer Serial analysis of geneexpression (SAGE) and subsequent microarray analysis previously revealedthat, compared with finite lifespan HMECs, cyclin D2 expression wassignificantly lower in a small panel of primary breast tumors (Nacht M,et al., Cancer Research 59:5464-5470 (1999). To confirm the validity ofthese findings, we investigated expression of cyclin D2 by RT-PCR inthree finite life span and 6 immortal HMECs, 11 breast cancer cell linesand 24 primary breast carcinomas. A ribosomal protein RNA, 36B4, wasco-amplified as an internal control. Abundant expression of cyclin D2mRNA was noted in all three finite life span HMECs and in 4 of 6immortalized HMECs. The two immortalized HMEC lines lacking cyclin D2expression were HBL100 and MCF10A. In contrast, 10 of 11 breast cancercell lines showed no detectable expression of cyclin D2. Only one breastcancer cell line, HS578T, expressed a low but detectable level of cyclinD2. Likewise, the results with primary tumors reflected the findings incultured cells. Eighteen of 24 primary breast carcinomas expressedsignificantly lower levels of cyclin D2 mRNA as compared with finitelifespan HMEC 184 and five other HMECs. As an additional control forcyclin D2 expression, the expression of cyclin D1 was analyzed in thesame panels of cell lines and tumors. Consistent with previousobservations Cyclin D1 mRNA was detectable in all the cell lines andprimary breast tumors tested. Thus, in both breast cancer cell lines andprimary tumors specific loss of cyclin D2, but not cyclin D1, mRNAexpression was observed.

Cyclin D2 mRNA expression in luminal and myoepithelial cells of thebreast It has been reported that cyclin D2 is expressed in myoepithelialbut not in luminal epithelial cells of the breast (Lukas J, (1995)).Therefore, lack of expression of cyclin D2 in breast cancers would beexpected, since the vast majority of these tumors originate from luminalrather than myoepithelial cells. This conclusion was based, however, onthe results from a single HMEC preparation. The present study used alarger panel of tissues. Luminal and myoepithelial cells isolated fromfour normal mammoplasty specimens from women aged 18 to 33 were used.Paired luminal and myoepithelial cells were obtained from the samebreast of two women. Each cell type was purified using immunomagneticbeads. The human luminal and myoepithelial cells were separated byvirtue of their exclusive expression of epithelial membrane antigen(EMA) and common acute lymphoblastic leukemia antigen (CALLA)respectively. The purity of the populations was checked byimmunocytochemistry using cytokeratins 18 and 19 as markers for luminalcells and cytokeratin 14 as a marker for myoepithelial cells. Thesetests showed that the final population was 95-99% pure in each case.Cyclin D2 expression was assessed in the purified cell preparations byRT-PCR. Cyclin D2 expression was observed in four of four purifiedluminal epithelial cells, as well as four of four myoepithelial cells.However, one luminal epithelial cell sample had a significantly lowerexpression of cyclin D2. Four HMECs of the 184 series, which stain forluminal cell markers- cytokeratins 8 and 18 and mucin, but not formyoepithelial cell marker- cytokeratin 14, also expressed cyclin D2mRNA. Thus, cyclin D2 mRNA was expressed in all eight of eight luminaland four of four myoepithelial cell preparations from the normal breast.

Western analysis reveals loss of cyclin D2 protein in primary tumors ForWestern blot analysis specific anti-cyclin D2 antibodies that did notcross-react with cyclin D1 were used. While cyclin D2 protein wasclearly detected in all seven HMECs tested (11-24, 1-26, 70N, 166372,81N, 9F1403 and 184A1), it was undetectable in the majority (10/13) ofprimary breast tumors. Thus, HMECs that were derived from normal breasttissue and expressed high levels of cyclin D2 mRNA show clearlydetectable levels of cyclin D2 protein as well. In contrast, primarybreast tumors that exhibited low or absent cyclin D2 mRNA showed acorresponding loss of the cyclin D2 protein.

The cyclin D2 promoter is hypermethylated in breast cancer cell linesand primary tumors In somatic cells, about 80% of the CGs aremethylated. Exceptions to this are the CpG islands in the promoterregion of many genes. CpG islands are GC-rich regions of DNA,approximately 1 kb in length, present in the promoters of more than 60%of human genes. Normally CpG islands are unmethylated and the chromatinin those sites is enriched in hyperacetylated histone and deficient inhistone H1, characteristic of active chromatin. Both unmethylated andmethylated DNA are assembled into nucleosomes.

The cyclin D2 promoter contains a CpG-rich region at 1000 to 1600base-pairs 5′ to the translation start site. To test whether aberrantmethylation is associated with loss of cyclin D2 expression, primers fora Methylation Specific PCR (MSP) assay were designed to rapidly screenfor cyclin D2 promoter methylation. Hypermethylation of the CpG richregion was detected in 11 of 11 breast cancer cell lines that alsolacked expression of cyclin D2 protein. Aberrant methylation was alsonoted in 49 of 106 (46%) primary breast carcinomas.

Next, to determine whether cyclin D2 promoter-methylation is atumor-specific phenomenon, DNA from histopathologically normal breasttissue adjacent to the surgically resected cancer was tested. All 11samples of normal breast epithelial tissue adjacent to carcinoma wereunmethylated at the CpG sites tested by MSP.

To further support the observation that cyclin D2 hypermethylation doesnot occur in normal HMECs and is associated with malignancy, normalbreast epithelial cells prepared by a variety of techniques wasexamined. By MSP analysis, cyclin D2 promoter was found to beunmethylated in seven mammary organoid preparations from reductionmammoplasties, and in five finite life span HMECs cultured fromnon-malignant breasts. The only exception to this finding was inimmortalized HMECs HBL100 and MCF10A, which contained hypermethylatedcyclin D2. As expected, these HMECs were the only two that did notexpress cyclin D2 mRNA.

To rule out the contribution of inflammatory blood cells present inbreast cancer specimens as the source of methylated cyclin D2, tensamples of peripheral blood cells (PBLs) from non-cancer patients weretested. All ten PBLs contained unmethylated cyclin D2 alleles.

Expression of cyclin D2 protein was undetectable in 10 of the 13 primarybreast cancers tested. However, methylation of the cyclin D2 promoterwas noted only in six of these ten primary tumors. This finding suggeststhat while methylation may cause silencing of cyclin D2 expression inmany breast cancers, alternative pathways account for the loss of theprotein in a proportion of these tumors.

Cyclin D2 promoter hypermethylation in preneoplasia Ductal carcinoma insitu (DCIS) is a preneoplastic lesion with a potential for progressionto invasive cancer. To determine if hypermethylation of the cyclin D2promoter occurs early in the evolution of breast cancer, MSP analysiswas performed on DNA from carefully microdissected samples of DCIS.Hypermethylation was noted in 44% of DCIS samples. In the cases whereadjacent invasive cancer was present as well, the methylation status ofboth lesions was concordant. This finding suggests that alteration ofcyclin D2 expression may be an early event, and may precedetransformation to the fully malignant stage of invasive carcinoma.

Re-expression of cyclin D2 mRNA in breast cancer cell lines Breastcancer cell lines MDAMB231 and MCF7 do not express cyclin D2 mRNA orprotein. If silencing of expression is mediated by promoter methylationand/or altered chromatin conformation, then demethylation of the gene byexposure to 5-aza 2′-deoxycytidine (5aza-dC), or treatment with thehistone deacetylase inhibitor, trichostatin A (TSA), should result inremoval of the repressive mechanism and re-expression of the gene.Indeed, when MDAMB231 and MCF7 cells were exposed to 5-aza-dC inculture, the cyclin D2 promoter was partially demethylated (as analyzedby MSP), and cyclin D2 mRNA expression was restored (as analyzed byRT-PCR). Further, exposure to TSA also led to re-expression of thecyclin D2 mRNA. These results suggest that methylation at the promoterregion plays a functional role in suppressing the expression of cyclinD2 in breast cancer.

Using RT-PCR, cyclin D2 expression was detected in four normal luminalepithelial cultures of the 184 series, in four of four purified luminalepithelial cell extracts, and in four of four myoepithelial cellextracts Using MSP, promoter hypermethylation was detected in 49/106(46%) of the tumors. Hypermethylation of the gene correlated with lackof cyclin D2 mRNA and/or protein expression. Thus, in about 50% ofbreast cancers, cyclin D2 silencing may be attributed to tumor-specificmethylation.

EXAMPLE 3 Hypermethylation and Loss of Expression of 14-3-3 Sigma

The extent of methylation of the cyclin 14-3-3 sigma-associated CpGislands in normal mammary epithelium, in breast cancer cell lines, andin primary mammary tumors, and expression of the 14.3.3 sigma mRNA andprotein in the same cells and tissues was examined.

Cell Lines and Tissues The breast cancer cell lines Hs578t, MDA-MB-231,MDA-MB-435 and MCF-7 and the human mammary epithelial cell lines,MCF-10A and HBL-100 were obtained and maintained according toinstructions (ATCC). The two matched tumor cell lines, 21PT and 21MTwere propagated as described (Band, et al. (1990) Cancer Res.50:7351-7357). Cultured normal human breast epithelial cell (HMEC)strains, 161, 184, 172, and 48, and the conditionally and fully immortalcell lines, 184A1(passage 15 and 99), and 185B5 were grown as described(http://www.lbl.gov/LBL-Programs/mrgs/review.html). Three additionalshort term cultures of HMECs,(#04372 and #16637) were grown according tospecifications (Clonetics). Primary breast tumor tissues were obtainedimmediately after surgical resection at the Johns Hopkins University orDuke University, and stored frozen at −80° C. Microscopic examination ofrepresentative tissue sections from each tumor revealed that thesesamples contained greater than 50% tumor cells. Microdissection ofprimary tumor cryosections was performed by using a laser capturemicroscope (Schutze, et al. (1998) Nat Biotechnol 16:737-42) or bymanually scraping the cells with a 20 G needle under 40× magnification(Umbricht, et al. (1999) Oncogene 18:3407-14.).

Northern Blot Analysis Total RNA was isolated from primary tumor tissuesusing Trizol Reagent (Life Technologies). Five micrograms were resolvedon 1.5% agarose/formaldehyde gels, and transferred to a nylon filterusing standard methods (Gene Screen, DuPont). A 375 bp cy-specific probewas generated using MCF-10A cDNA as a template and the primers5′-ACAGGGGAACTTTATTGAGAGG-3′ (SEQ ID NO:25) and5′-AAGGGCTCCGTGGAGAGGG-3′ (SEQ ID NO:26). Hybridizations were done inQuikhyb (Stratagene) according to the manufacturer's instructions.Filters were exposed to autoradiographic film for up to 5 days. To testfor uniform loading of the samples, blots were stripped and reprobedwith a 1.5 kb DNA fragment specific for 18S rRNA (ATCC, Clone #HHCSA65).

Loss of Heterozygosity (LOH) Studies A TG repeat sequence in the 3′UTRof a was amplified using: 5′-GAGGAGTGTCCCGCCTTGTGG-3′ (sense; SEQ IDNO:27) and 5′-GTCTCGGTCTTGCACTGGC-3′ (antisense; SEQ ID NO:28) primers,which yields a product of 117 bp. The 25 μl reactions contained 50 ng oftemplate DNA (10), 17 mM NH₄SO₄, 67 mM TrisCl (pH 8.8), 6.7 mM MgCl₂, 1%DMSO, 1.5 mM dNTP, 20 ng of each primer, 2 ng of γ-³²P-labeled senseprimer, and 0.5 μl Taq polymerase. PCR conditions were as follows: 1cycle of 94° C. for 90 s; 35 cycles of 94° C. for 1 min, 57° C. for 30s, 72° C. for 30 s; and 1 cycle of 72° C. for 5 min. PCR products werefractionated on a sequencing gel, which was exposed to autoradiographicfilm overnight (Evron, et al. (1997) Cancer Res, 57:2888-9).

Mutation Analysis A 1.2 kb PCR product, encompassing the entire a codingsequence, was generated using two primers, 5′-GTGTGTCCCCAGAGCCATGG-3′(sense; SEQ ID NO:29) and 5′-GTCTCGGTCTTGCACTGGCG-3′ (antisense; SEQ IDNO:30). The PCR reaction contained 50 ng of DNA, 6.4% DMSO, 1.5 mMdNTPs, 100 ng of each primer and 0.5 μl Taq polymerase in a 50 μlreaction volume. α-³³P cycle sequencing was performed using theAmplicycle sequencing kit (Perkin Elmer). Four different α-³³P-labeledprimers were used to sequence the entire a coding sequence: (antisense;SEQ ID NO:31) 5′-CACCTTCTCCCGGTACTCACG-3′, (sense; SEQ ID NO:32)5′-GAGCTCTCCTGCGAAGAG-3′, (sense; SEQ ID NO:33)5′-GAGGAGGCCATCCTCTCTGGC-3′ and (antisense; SEQ ID NO:34)5′-TCCACAGTGTCAGGTTGTCTCG-3′.

Transfection of Human Breast Cancer Cell Lines 1.5×10₅ of MCF-7,MDA-MB-231, and Hs578t, or 2.5×10₅ cells of MDA-MB-435 breast cancercells were seeded in six-well plates. The following day, transfectionswere performed using Trans IT-LT1 (Mirus Corp.) as per manufacturer'sinstructions. Plasmids used in the transient transfections include: KKHluciferase, containing 4 kb of the cy-promoter linked to the luciferasegene in the pGL3-Basic vector (Promega); pCMV-β-gal (Clontech), whichwas used to correct for the efficiency of transfection; and pGL3-Basic(Promega), which was used as a negative vector control against which KKHluciferase activities were compared. Two μg of luciferase reporterplasmid or the pGL3-Basic vector control and 0.5 μg of CMV-β-galreporter plasmid were used for each transfection.

Luciferase and β-galactosidase Assays Cell lysates were madeapproximately 48 hr post-transfection as per manufacturer's instructions(Promega, Luciferase Assay System). Luciferase and β-galactosidaseactivities were quantitated using the luciferase assay system (Promega)and the Aurora GAL-XE® reporter gene assay (ICN Pharmaceuticals, Inc),respectively. Experiments were done in triplicate. Luciferase activitywas first normalized for efficiency of transfection by using the ratioof luciferase to β-galactosidase activity. For each transfected cellline, the results were compared with the mean of pGL3 vector controllevels and expressed as fold elevated expression above pGL3. The meansand standard deviations of the results of all experiments werecalculated.

Sodium Bisulfite DNA Sequencing Genomic DNA was subjected to sodiumbisulfite modification as described in Herman et al. ((1996) Proc. Natl.Acad. Sci. USA, 93:9821-9826). Bisulfite-converted DNA was amplified, asdescribed above, using primers that encompass the first exon of the σgene: 5′-GAGAGAGTTAGTTTGATTTAGAAG-3′ (sense primer with start at nt8641; SEQ ID NO:35) and 5′-CTT ACTAATATCCATAACCTCC-3′ (antisense primerwith start at nt 9114; SEQ ID NO:36) which generated a 474 bp PCRproduct. Conditions for PCR were as follows: 1 cycle at 95° C. for 5min; 35 cycles at 95° C. for 45 s, 55° C. for 45 s and 72° C. for 60 s;and 1 cycle at 72° C. for 4 min. The product was purified using a QiagenPCR purification kit (Qiagen Corp) and sequenced using the sense primerwith an ABI automated fluorescent sequencer according to themanufacturer's instructions.

Methylation-specific PCR (MSP) One μg genomic DNA was treated withsodium bisulfite as described in (Herman, et al. (1996) Proc. Natl.Acad. Sci. USA 93, 9821-9826), and was analyzed by MSP using a primerset that covered CG dinucleotide numbers 3, 4, 8 and 9. Primers specificfor methylated DNA: 5′-TGGTAGTTTTTATGAAAGGCGTC-3′ (sense; SEQ ID NO:37)and 5′-CCTCTAACCGCCCACCACG-3′ (antisense; SEQ ID NO:38), and primersspecific for unmethylated DNA: 5′-ATGGTAGTTTTTATGAAAGGTGTT-3′ (sense;SEQ ID NO:39) and 5′-CCCTCTAACCACCCACCACA-3′(antisense; SEQ ID NO:40)yielded a 105-107 bp PCR product. The PCR conditions were as follows: 1cycle of 95° C. for 5 min; 31 cycles of 95° C. for 45 s, 56° C. for 30 sand 72° C. for 30 s; and 1 cycle of 72° C. for 4 min.

Treatment of Cells with 5′-aza-2′-deoxycytidine (5-aza-dC) Cells wereseeded at a density of 2×10⁶ cells per 100-mm plate. 24 h later cellswere treated with 0.75 μM 5-aza-dC (Sigma) (Ferguson, et al. (1995)Cancer Res 55:2279-2283). Total cellular RNA and genomic DNA wereisolated from the cells at 0 and 3 days after addition of 5-aza-dC asdescribed herein.

RT-PCR RNA was treated with RNase-free DNAse (Boehringer-Mannheim) (1μg/μl) for 2 h at 37° C., followed by heat inactivation at 65° C. for 10min. RT reactions contained 1 μg DNAse treated RNA, 0.25 μg/μl pdN6random primers (Pharmacia), 1× first strand buffer (GibcoBRL), 0.5 mMdNTP (Pharmacia), and 200 U MMLV-RT (GibcoBRL), and were incubated for 1h at 37° C. PCR was performed using the σ-specific primers5′-GTGTGTCCCCAGAGCCATGG-3′ (SEQ ID NO:41) and 5′-ACCTTCTCCCGGTACTCACG-3′(SEQ ID NO:42) using buffer conditions described herein. The PCRconditions were: 1 cycle of 95° C. for 5 min; 30 cycles of 60° C. for 45s, 72° C. for 45 s and 95° C. for 45 s. PCR samples were resolved byelectrophoresis in a 2% agarose gel.

Assay for G1 and G2 checkpoint and chromosomal aberrations The G1 cellcycle checkpoint and chromosomal aberrations in mitosis were assessed asdescribed previously (Pandita, et al. (1996) Oncogene 13:1423-1430).Specifically, cells in plateau phase were irradiated with 3 Gy,sub-cultured after 24 h, and metaphases were collected. G1 typeaberrations were examined at metaphase. All categories of asyrmnetricchromosome aberrations were scored: dicentrics, centric rings,interstitial deletions/acentric rings, and terminal deletions.

The efficiency of G2 checkpoint control was evaluated by measuring theproportion of cells in metaphase after irradiation. Chromosomalaberrations at mitosis were assessed by counting chromatid breaks andgaps per metaphase as described elsewhere (Morgan, et al. (1997) MolCell Biol 17:2020-2029). Specifically, cells in exponential growth phasewere irradiated with 1 Gy. Metaphases were harvested 45 and 90 minutesfollowing irradiation and examined for chromatid type breaks and gaps.Fifty metaphases each were scored for G1 and G2 types of chromosomalaberrations.

Introduction of σ into the σ-negative breast cancer cell line MDA-MB-435by adenoviral infection Cells were seeded and grown to 50% confluence.Adenovirus encoding either σ or β-galactosidase (Hermeking, et al.(1997) Mol. Cell 1:3-11) was added to the culture at a multiplicity ofinfection of 5000:1 and infection was allowed to take place overnight.The cells were harvested, fixed and stained with Hoechst dye andsubjected to FACS analysis.

σ Expression in Normal, Immortalized and Tumorigenic Breast EpithelialCells By SAGE analysis, σ was found to be expressed at an average of7-fold lower levels in three human breast cancer cell lines, 21PT, 21MTand MDA-MB-468 than in two populations of normal human mammaryepithelial cells (HMEC). Northern blot analysis was performed to confirmthis finding in other breast cancer cell lines and in primary breasttumors. No expression of σ was detected in 45 of 48 (94%) primarytumors. In contrast, σ was expressed at easily detectable levels in all6 cultured HMEC populations and 5 immortalized but nontumorigenic celllines. These results indicate that loss of σ gene expression is afrequent event in human breast cancer.

Genetic alterations within the σ gene Possible causes for loss of σ geneexpression in breast tumors include deletion of the chromosomal regioncontaining the gene or intragenic mutations that lead to decreased mRNAstability. σ localizes to chromosome 1p35, an arm that has beenextensively studied for LOH in breast cancer (Hermeking, et al. (1997)Mol. Cell 1, 3-Bieche, et al. (1995) Genes Chrom. Cancer 14:227-251).LOH has been observed for the 1p32-36 region at a frequency of 15-25%.However, it is not known whether the region lost in these tumorsincludes σ (Genuardi, et al. (1989) Am. J. Hum. Genet. 45:73-82; Trent,et al. (1993) Genes Chrom Cancer 7:194-203; Nagai, et al. (1995) CancerRes. 55:1752-1757; Tsukamoto, et al. (1998) Cancer 82:317-322).Therefore, the loss of σ by utilizing a TG repeat sequence within the 3′UTR of the C gene itself was examined. Using primers that span the TGrepeats, the locus in 45 sets of normal and tumor DNA pairs was studied.Twenty of 45 (44%) of the patients were found to be heterozygous withrespect to the length of the PCR-product. Only one of the 20 tumorspecimens exhibited LOH (Table 2). Eleven of these 20 samples weretested by Northern blot analysis, and no σ transcripts were detectable.These results prompted an examination whether there were smaller geneticchanges within the coding region of σ. The entire 1190 bp coding regionfrom σ-nonexpresssing (σ-negative) breast cancer cell lines, MDA-MB-435and Hs578t and 7 primary tumor tissues was amplified with PCR andsequenced. No mutations were found. In addition, 25 primary tumor DNAsamples were analyzed by single stranded conformation polymorphism, andno abnormalities were detected. These results suggest that geneticalterations within σ are not a primary mechanism for loss of geneexpression. TABLE 2 Incidence of σ alterations in breast cancer No. withσ expression, No. with methylated LOH/total, No. with Northern blotσσ/total TG repeat mutation/total Sample analysis Sequencing MSP PCRSequencing SSCP Normal breast Mortal HMEC strains 6/6 01 0/3 ImmortalHMEC lines 5/5 01 0/5 Reduction 0/6 01 mammoplasty, microdissectedepithelium Breast cancer Cell Lines MCF-7 +

MDA-MB-231 +

MDA-MB-435

+ +

Hs578t

+ +

Primary tumors  2/45 10/10 43/50  1/20 0/7  0/25 MICRODISSECTED 32/32CARINOMA

Epigenetic alterations of the σ gene Next tested was whether the lack ofσ mRNA was due to deficiencies in factors required for σ transcription.The two breast cancer cell lines, MDA -MB-435 and Hs578t, served asmodel systems for σ-negative primary tumors that harbored wild type σalleles, while the two breast cancer cell lines, MCF-7 and MDA-MB-231,served as σ-positive controls since they both express detectable levelsof σ. The plasmid KKH-luciferase contains 4 kb of sequence upstream ofthe transcriptional start site of σ linked to the luciferase reportergene; this upstream region contains the sequences necessary for p53 andγ-irradiation-inducible transcription of σ (5). Following transienttransfection of the four cell lines with the reporter plasmid, highlevels of expression was observed (70- to 300-fold above thepromoterless parental vector) in both σ-negative and σ-positive breastcancer cell lines. These results indicate that the σ-negative breastcancer cells, like the σ-positive cells, are able to supporttranscription from the a promoter equally well, and contained factorsrequired for transcription.

σ has a CpG rich region (CpG island) within its first and only exon thatbegins near the transcription initiation site and ends approximately 800bp downstream. To explore a role of hypermethylation in silencing a geneexpression, the nucleotide sequence of this region was determined aftertreating the DNA with sodium bisulfite (Frommer, et al. (1992) Proc.NatL. Acad. Sci. USA 89:1827-1831). PCR primers were designed to amplifya region spanning 27 CpG dinucleotides within the CpG island. Nosignificant methylation was observed using DNAs from four σ-positivecell lines including 2 HMECs (184, MCF-10A) and two tumorigenic breastcancer cell lines (MCF-7 and MDA-MB-231). In contrast, DNAs from twoσ-negative breast cancer cell lines, HS578t and MDA-MB-435, were fullymethylated at all of the CpG sites. Since there was a strong correlationbetween σ-methylation status and mRNA expression in all the cell linesexamined, 10 σ-negative primary breast tumors were also examined. All ofthe tumor DNAs exhibited partial or complete methylation of the 27 CpGdinucleotides.

Next, an MSP assay was utilized to detect methylation of the CpG island,using primers spanning the region between CpG dinucleotides 3 and 9within the σ gene. Primers were designed that take advantage of thenucleotide sequence differences between methylated and unmethylated DNAas a result of bisulfite modification. By this method, 5/5 σ-positiveHMEC strains were completely unmethylated. In addition, DNAs from theσ-positive immortalized breast epithelial cells (MCF-10A, HBL-100) andbreast cancer cell lines (MCF-7 and MDA-MB-231), were also unmethylatedat the sites examined. In contrast, DNAs from the σ-negative breastcancer cell lines, Hs578t and MDA-MB-435, were fully methylated.Similarly, 43 of 50 samples from primary breast tumors were partially orcompletely methylated. Of these 43 tumors, 26 were examined by Northernblot analysis, and all 26 lacked detectable σ gene expression. Three ofthe seven unmethylated breast tumor samples also lacked σ transcripts;the expression pattern for the remainder was not tested. These resultsdemonstrate that aberrant methylation of σ is a frequent event in breastcancer, but that other mechanisms are responsible for silencing the genein a small fraction of breast tumors.

Previous reports indicate that a gene expression is restricted todifferentiated epithelial cells. In order to clearly ascertain thecellular origin of methylated DNA, normal and tumor tissues weremicrodissected and analyzed for σ methylation by MSP. All six DNAsamples of microdissected mammary epithelial cells obtained fromreduction mammoplasty specimens were unmethylated. In contrast, all 32samples of DNA from microdissected breast carcinomas were methylatedwithin the a CpG island. These results indicate that hypermethylation ofthe σ gene is associated with loss of gene expression in the majority ofprimary breast tumors. The data from gene expression, genetic andepigenetic studies are summarized in Table2.

In order to determine the effect of methylation on a gene expression,two fully methylated, σ-negative cell lines, Hs578t and MDA-MB-435, weretreated with the DNA methyltransferase inhibitor, 5-aza-dC. Treatment ofcells with 0.75 μM 5-aza-dC for 3 days led to demethylation of the CpGrich region encompassed by the MSP primers. Moreover, 5-aza-dC treatmentresulted in reactivation of gene expression, as demonstrated by RT-PCR.These results demonstrate that methylation is at least partiallyresponsible for loss of σ transcription in breast cancer cells.

Functional consequences of loss of σ in breast cancer cells The functionof human σ has been analyzed in human colon carcinoma cells. Thesestudies demonstrated that following ionizing irradiation, a sequesterscdc2-cyclin B1 complexes in the cytoplasm, thus arresting the cells inG2. These actions prevent the cell from initiating mitosis before repairof its damaged DNA. Colon carcinoma cells lacking σ can still initiate,but do not maintain, G2 arrest, leading to mitotic catastrophe and celldeath.

In an attempt to determine the effects of loss of σ gene expression oncell cycle regulation in breast cancer cells, the effects ofγ-irradiation on the σ-negative breast cancer cell lines, MDA-MB-435,21NT, and 21MT, and the σ-positive breast cancer cell line, MCF-7 weretested. First, G1 type chromosomal aberrations were examined 24 h aftercells were exposed to 3 Gy of γ-irradiation. All categories of G1-typechromosomal aberrations were scored at metaphase; their frequency wasidentical in the two cell types. These results indicate that theexamined cell lines have similar G1 cell cycle checkpoint controlresponses to ionizing radiation.

Next, the G2 checkpoint function in the four cell lines was evaluated.Cells in exponential growth phase were γ-irradiated with 1 Gy andmetaphases were examined for chromatid type breaks and gaps. DefectiveG2 arrest will increase these values. The results show a strikingdifference in the ability of σ-negative cells and σ-positive cells torepair their damaged DNA. Forty-five minutes post irradiation,σ-negative cells exhibited up to twice as many G2 type chromosomalaberrations as MCF-7 cells. This number increases to three-fold by 90minutes. Moreover, while repair of DNA damage was evident in the MCF-7cells, as evidenced by a decrease in the number of G2 type aberrationsbetween 45 and 90 minutes, no decrease was seen in σ-negative cells.

Finally, in order to further demonstrate the role of σ in G2 checkpointfunction in breast cells, a cloned copy of the gene was overexpressed inthe σ-negative cell line MDA-MB-435 as well as in normal breastepithelial cells using the adenovirus expression system used to expressσ in colon cancer cells (5). Overexpression of σ in these breastepithelial cells led to a rapid and permanent G2 arrest, whereas thecontrol virus-infected cells showed no effect. These results indicatethat although the σ-negative cell lines have a functional G1 cell cyclecheckpoint, they accumulate more genetic damage following irradiation,which is consistent with its failure to arrest in G2 in response to DNAdamage.

In summary, these results show that in striking contrast to normalbreast tissue, greater than 90% of breast cancers lack detectableexpression of σ. Hypermethylation of the σ gene occurs in a CpG-richregion that extends from the transcriptional initiation site to themiddle of the coding region. Bisulfite genomic sequencing of this 500 bpregion showed that is consistently and densely methylated in σ-negativecell lines and primary breast tumors. Several studies have clearlydocumented that gene activity correlates inversely with the density ofgene-specific CpG island methylation, but is less dependent on theposition and distance of the methylated DNA sequences from thetranscriptional initiation site. With respect to a, dense methylationjust downstream of its transcriptional start site is strongly associatedwith gene silencing. Furthermore, in σ-negative cell lines,5-aza-dC-induced demethylation of the CpG island leads to reactivationof gene expression, indicating that hypermethylation plays a causal rolein a gene inactivity.

EXAMPLE 4 Hypermethylation and Loss of Expression of RAR B2

The extent of methylation of the RAR β2-associated CpG islands in normalmammary epithelium, in breast cancer cell lines, and in primary mammarytumors, and expression of the RAR β2 mRNA and protein in the same cellsand tissues was examined.

Cell cultures Human epithelial mammary cells (HEMC) from reductionmammoplasty including three mortal strains, 184, 48R and 172R, and twoimmortal strains, 184A1 and 184B5, were obtained and cultured accordingto the protocols designed by Dr. Martha Stampfer (see the HMEC Homepage,http://www.lbl.gov/*mrgs/index.htlm) using Clonetics (Walkersville, Md.,USA) reagents. Human breast cancer cell lines were maintained inDulbecco's modified Eagle's medium (GIBCO) (Hs578t,MCF-7, MDA-MB-231 andT47D) or IMEM medium (Biofluids) (MDA-MB-435, MDA-MB-468, ZR751) with 5%fetal calf serum (FCS). For drug treatments, exponentially growing cellswere seeded in 10 cm² plates at a density of 36105 cells/plate or in6-well plates at 16105 cells/well. Cells were allowed to attachovernight before the addition of the appropriate concentration of5-Aza-2′ deoxycytidine (5-Aza-CdR) (Sigma), Trichostatin A (TSA) (Sigma)or RA(Sigma). When reduction of retinoids was required, cells weretreated in either medium with 0.5% FCS or charcoal-dextran stripped FCS(Hyclone). At the indicated time points, both attached and detachedcells were harvested, counted with Trypan Blue (Life Technologies) andprocessed for DNA or RNA extraction. 5-Aza-CdR was dissolved in 0.45%NaCl containing 10 mM sodium phosphate (pH 6.8). Trichostatin A andall-trans-retinoic acid (RA) (Sigma) were reconstituted in absoluteethanol (solvent). The growth inhibition (%) was calculated as:(1-NT/NC)6100, where NT is the number of treated cells and NC is thenumber of control cells.

Tissue samples Normal and tumor tissues were collected from existingtumor banks (Instituto per lo Studio e la Cura dei Tumori, Milan; theCancer Center, Rotterdam, the Johns Hopkins Breast Cancer Program,Baltimore, Md., USA). All tumor samples were obtained from excessclinical specimens and institutional guidelines for the acquisition andmaintenance of such specimens were followed. DNA and RNA extraction:Extraction of DNA and RNA from breast cancer cell lines was performed byusing DNAzol and Trizol respectively (LifeTechnologies) according to themanufacturer's instructions. Genomic DNA was further treated with 500mg/ml proteinase K at 55° C., extracted with phenol-chloroform-isoamylicalcohol (24:24:1) (CIA) and ethanol precipitated. Extraction of DNA fromparaffmated breast cancer and lymph node tissues was essentiallyperformed as previously described (Formantici et al., 1999). One tothree consecutive sections estimated to contain at least 90% tumor cellswere incubated at 58° C. overnight in 200 ml of extraction buffer (50 mMKCl, 10 mM Tris-HCl (pH 7.5), 2.5 mM MgCl₂, 0.1 mg/ml gelatin,0.45%NP-40, 0.45% Tween 20, and the solution was heated at 95° C. for 15 minto inactivate the proteinase K and then centrifuged at 6000 r.p.m. TheDNA in the supernatant was used for analysis.

Southern blotting Genomic DNA (7 mg) was digested overnight with 15 U/mgof XbaI, HpaII and MspI enzymes, electrophoresis on a 0.8% agarose geland transferred to Hybond-N filter. A 227 bp probe was amplified usingthe sense 5′-AGA GTT TGA TGG AGTTGG GTG GAG-3′ (SEQ ID NO:43) andantisense 5-CAT TCG GTT TGGGTC AAT CCA CTG-3′ (SEQ ID NO:44) primers,gel purified and labeled with ³²P-dCTP using the Megaprime DNA labelingsystem (Amersham). After hybridization the filters were washed andexposed to X-ray film at −80° C. for autoradiography.

Methylation specific PCR (MSP) Bisulfite modification of genomic DNA wasessentially performed as described by Herman et al. (1996) and describedherein. Modified DNA was used immediately or stored in aliquots at −20°C. The PCR mixture contained 1× PCR buffer (16.6 mM ammonium sulfate, 67mM Tris (pH 8.7), 1.5 mM MgCl₂), dNTPs (each at 1.25 mM), primers (300ng each per reaction),and bisulfite-modified DNA (50 ng) or unmodifiedDNA(50 ng). Reactions were hot started at 95° C. before the addition of2.5 U of Taq polymerase (Qiagen). Amplification was carried out in aThermal Cycler 480 Perkin Elmer for 30cycles (1 min at 94° C., 1 min atthe annealing temperature (at) selected for each primer pair, 1 min at72° C.), followed by 4 min at 72° C. Twelve μl of the PCR reaction wereelectrophoresed onto 1.5% agarose gels, stained with ethidium bromideand visualized under UV. Two primer pairs, W3 sense5′-CAGCCCGGGTAGGGTTCACC-3′ (SEQ ID NO:45), W3 antisense5′-CCGGATCCTACCCCGACGG-3′ (SEQ ID NO:46), and W4sense5′-CCGAGAACGCGAGCGATCC-3′ (SEQ ID NO:47) and W4 anti-sense5′-GGCCAATCCAGCCGGGGCG-3′ (SEQ ID NO:48), were designed on the human RARβ2 sequence (Shen et al., 1991) and used to control the Na bisulfitemodification. The primer pairs selected to detect the unmethylated DNAwere as follows: U1sense 5′-GTG GGT GTA GGT GGA ATA TT-3′ (SEQ ID NO:49)and Ulantisense 5′-AAC AAA CAC ACA AAC CAA CA-3′ (SEQ ID NO:50) (at 55°C.); U2 sense 5′-TGT GAG TTA GGA GTA GTG TTTT-3′ (SEQ ID NO:51) and U2antisense 5′-TTC AAT AAA CCC TAC CCA-3′ (SEQ ID NO:52) (at 49° C.); U3sense 5′-TTA GTA GTT TGG GTA GGGTTT ATT-3′ (SEQ ID NO:53) and U3antisense 5′-CCA AAT CCT ACC CCAACA-3′ (SEQ ID NO:54) (at 55° C.); U4sense 5′-GAT GTT GAG AAT GTGAGT GAT TT-3′ (SEQ ID NO:55) and U4antisense 5′-AAC CAA TCC AACCAA AAC A-3′ (SEQ ID NO:56) (at 55° C.); Thesequences of the primers to detect the methylated DNA were: M1 sense5′-AGC GGGCGT AGG CGG AAT ATC-3′ (SEQ ID NO:57) and M1 antisense5′-CAACGA ACG CAC AAA CCG ACG-3′ (SEQ ID NO:58) (at 63° C.); M2 sense5′-CGT GAG TTA GGA GTA GCG TTT C-3′ (SEQ ID NO:59) and M2 antisense5′-CTT TCG ATA AAC CCT ACC CG-3′ (SEQ ID NO:60) (at 57° C.); M3 sense5′-GGT TAG TAG TTC GGG TAG GGTTTA TC-3′ (SEQ ID NO:61) and M3 antisense5′-CCG AAT CCT ACC CCGACG-3′ (SEQ ID NO:62) (at 64° C.); M4 sense 5′-GTCGAG AAC GCG AGCGAT TC-3′ (SEQ ID NO:63) and M4 antisense 5′-CGA CCA ATCCAA CCGAAA CG-3′ (SEQ ID NO:64) (at 64° C.).

M and U primers were designed in the same regions, with one or twonucleotide differences to meet annealing requirements. Fragment M3(position 773±1007) contains the βRARE (792±808) and the transcriptionstart site (position 844);fragment M4 (position 949±1096) contains anSp1 element (position 1074±1081).

RT±PCR The exon 5 (sense primer 5′-GAC TGT ATG GAT GTTCTG TCA G-3′; SEQID NO:65) and exon 6 (antisense primer 5′-ATT TGTCCT GGC AGA CGA AGCA-3′; SEQ ID NO:66) were designed on the basis of published RAR β2transcript (de The' et al., 1990;van der Leede et al., 1992) and used toamplify 50 ng of DNase treated total RNA using the SuperscriptOne-StepRTt PCR System (Life Technologies). RT±PCR with actin primers(sense primer 5′-ACC ATG GAT GAT GAT ATCG-3′; SEQ ID NO:67 and antisenseprimer 5′-ACA TGG CTG GGG TGTTGA AG-3′; SEQ ID NO:68) was used as aninternal RNA control.

The RAR β2 promoter is methylated in breast cancer cell linesindependently of their ER status and RA-inducibility RAR transcriptionwas first tested in a panel of breast cancer cell lines grown in theabsence of exogenous RA, by reverse transcriptase-PCR (RT±PCR), usingprimers encompassing exons 5 and 6 (de The' et al.,1990; van der Leedeet al., 1992; Toulouse et al., 1997). Under these conditions, only onecell line, Hs578t, produced a detectable 256 bp RT±PCR product. Thus,previous reports were confirmed that RAR β gene expression is downregulated/lost in breast cancer cell lines. Growing cells in thepresence of RA can assess the distinction between down regulation andloss. As previously reported (Swisshelm et al., 1994; Liu et al., 1997;Shang et al., 1999), we observed induction of RAR β expression andgrowth inhibition inT47D, MDA-MB-435, MCF7 and ZR75-1 cell lines treatedfor 48 h with 1 μM RA, but not in the MDA-MB-231 and MDA-MB-468 celllines.

To see whether the RAR β2 methylation status correlated with the ERstatus, the methylation status was examined at RAR β2 in a panel ofER-positive (MCF7,T47D, ZR75-1) and ER-negative (Hs578t, MDA-MB-231,MDA-MB-435, MDA-MB-468) cell lines.

By Southern blotting, the CpG island of the RAR β2 promoter within a 7.5kb XbaI DNA fragment encompassing the TATA box, the βRARE, thetranscriptional start site (TS) and the 5′ untranslated region of exon 5was examined. In this region nine HpaII sites can be identified (Shen etal., 1991; Baust et al.,1996). The DNA methylation status was analyzedby using the methylation-sensitive enzyme, HpaII. MspI, the isoschizomerof HpaII, insensitive to methylation, was used as a positive control.The PCR probe spans the βRARE and the TATA box regions. The same 7.5 kbregion was previously analyzed in a colon carcinoma cell line, and thesize of all the possible fragments relative to the most 3'HpaII sitewere reported (Cote′ and Momparler, 1997). Genomic DNA from theER-positive, RA-inducible cell line T47D is digested to completion,indicating that it is not methylated at any of the HpaII sites. Incontrast, DNA from the ER-positive, RA-inducible ZR75-1 cell line andDNA from the ER-negative, RA-resistant MDA-MB-231 cell line showed to bedifferentially methylated at the methylation-sensitive sites. Usingmethylation-specific PCR (MSP), we further analyzed a 616 bp long RAR β2region from nucleotide 481 to nucleotide 1096 (Shen et al., 1991) in allthe cell lines. MSP entails the modification of genomic DNA by sodiumbisulfite that converts all unmethylated, but not methylated, cytosineto uracil (Herman et al., 1996). The genomic DNAs from four breastcancer cell lines ZR751,MCF7, MDA-MB-231, MDA-MB-468 showed partial tocomplete methylation of the promoter region. The human mammaryepithelial cell (HMEC) strain48R, expressing RAR β and three breastcancer cell lines, the RAR β-positive Hs578t and the RA-inducibleMDA-MB-435 and T47D, revealed only the (U) unmethylated PCR products.

These results indicate that hypermethylation of the RAR β2 promoteroccurs in breast cancer cell lines irrespective of the ER status, andcan be detected in both RA-inducible, and RA-resistant breast cancercells.

RAR β2 is unmethylated in both mortal and immortalized HMEC, but ismethylated in primary breast tumors The next question examined waswhether hypermethylation of RAR β2 promoter in cell lines has correlatesin clinical breast cancer. As a normal control, the HMEC mortal strains(48R, 172R), that are the closest representation of normal mammaryepithelial cells available were examined. Also analyzed were twoimmortal mammary epithelial strains (184A1 and 184B5). The DNA of thesestrains was found to be unmethylated. Consequently, methylation of RARβ2 may be an event in the progression of breast cancer, followingimmortalization. Genomic DNAs from three paraffinated samples of breasttumors, two ER-positive (T1, T2)and one ER-negative (T3), estimated tocontain more than 90% tumor cells, were analyzed with all MSP primerpairs, and shown to be partially methylated. Both microdissected breaststroma, and microdissected normal epithelial cells were foundunmethylated at RAR β2, making it very likely that the U products in thetumor samples were amplified either from residual normal epithelialcells, or stromal cells mixed to tumor cells. DNAs from matchinghistologically tumor free lymph node samples (N1±N3), were similarlyanalyzed and produced only the unmethylated PCR products. The DNA ofadditional 21 tumors was performed using two sets of primer pairs (U3/M3andU4/M4). Fifteen (7 ER-positive and 8 ER-negative) of the 24 tumorspresented methylation at the RAR β2promoter. With the same primer setshypermethylation at RAR β2 was detected in the DNA of ten out of 39primary breast tumors collected, and analyzed independently, at theJohns Hopkins University. The overall data indicate thathypermethylation at RAR β2 promoter occurs in approximately one third ofprimary breast tumors, and that the RAR β2 methylation state isindependent of the ER status of the tumor.

5-Aza-CdR induces partial demethylation at the RAR β2CpG island andreactivation of RAR β gene expression In order to determine whether DNAmethylation is affecting, at least in part, RAR β gene expression, allcell lines showing methylation at the RAR β2 promoter were treated withthe DNA methyl transferase inhibitor, 5-Aza-CdR. Treatment of cells witheither0.4 or 0.8 mM 5-Aza-CdR for 3 days, led to partial demethylationof the CpG rich RAR β2 region. This was evident both by Southernanalysis in the MDA-MB-231cell line, and by MSP in all cell lines.Moreover, 5-Aza-CdR treatment resulted in reactivation of geneexpression both in RA-inducible MCF7 and ZR75-1, and RA-resistantMDA-MB-231 and MDA-MB-468 cells. Subsequent studies examined whetherreactivation of RAR β expression by 5-Aza-CdRA-resistant cells could beenhanced by RA. Using non-quantitative RT±PCR, a difference could notappreciated in the level of RAR β transcription in MDA-MB-231 cellstreated with 0.4 mM5-Aza-CdR alone, or in combination, with 1 μM RA. Inthis experiment, 5-Aza-CdR alone, or in combination with RA, produced 63and 96% growth inhibition respectively. In the same experiment,treatment with 1 μM RA alone produced a negligible effect on growthinhibition (52%). A synergistic effect of the two drugs on cancer cellswas previously reported (Cote' and Momparler,1997; Bovenzi et al.,1999).

These data indicate that DNA methylation is, at least, one factorinfluencing the down regulation/loss of RAR β transcription in breastcancer cell lines with a methylated RAR β2 promoter. Cells treated with5-Aza-CdR alone, or in combination with RA, showed re-expression of RARb, which may have contributed, along with the toxic5-Aza-CdR, to theobserved growth inhibition.

The HDAC inhibitor TSA can reactivate RAR P expression in RA-resistantcells; demethylation of the RAR β2 promoter is not an absoluterequirement for RAR β reactivation The chromatin status at a given locuscan be dynamically influenced by the degree of acetylation/deacetylationdue to HAT/HDAC activities. Absence of RAR β regulatory factors, likeRAR a, as well as DNA-methylation, can contribute to pattern chromatinmodifications at RAR β promoter in RA-resistant cell lines. One of thesecell lines, MDA-MB-231, lacks RA-inducible RARα activity (Shao et al.,1994) and displays a RAR β2 methylated promoter. A subsequent study wasdesigned to probe indirectly whether the level of HDAC at RAR β2 caninfluence RAR β expression, by testing the effect of TSA, a HDACinhibitor on MDA-MB-231cells (Yoshida et al., 1995). Cells were treatedfor 2 days, in the presence or absence of 100 ng/ml TSA alone, or incombination, with 1 μM RA. By using RT±PCR, it was clear that, unlikecells treated with RA alone, cells treated with a combination of RA andTSA re-expressed RAR β mRNA. Under the same experimental conditions, 100ng/ml TSA alone, or in combination with 1 mM RA, produced 77and 92%growth inhibition, respectively. Treatment with 1 μM RA alone did notaffect significantly growth inhibition (52%). By MSP analysis, it wasassessed that RAR β expression was restored in the presence of amethylated RAR β2 promoter. This finding indirectly shows that globalalterations of HDAC activity, generated by TSA in MDA-MB-231 cells,involved RAR β2 resulting in RA-induced RAR β expression. Further,demethylation at RAR β2 did not seem to be an absolute requirement forRAR 1 gene expression inMDA-MB-231 cells. Noteworthy, persistence ofmethylation at RAR β2 was observed also in MCF7 cells where RAR βtranscription could be restored in the presence of RA. Growth inhibitionwas observed in cells treated with TSA alone, or in combination, withRA. Very likely, RAR β along with TSA, a drug known to induce growthinhibition(Yoshida et al., 1995), contributed to the massive growthinhibitory effect that was observed.

These results show that RAR β2 promoter is methylated in breast cancer.This study presents evidence that, in breast cancer cells, RAR β2promoter undergoes DNA hypermethylation, an epigenetic change known toinduce chromatin modifications and influence gene expression.Methylation of the RAR β2 promoter region was detected, both in breastcarcinoma cell lines, and a significant proportion of primary breasttumors. RAR β2 methylation status did not correlate with the ER statusof breast cancer cells and was observed both in in situ lesions andinvasive tumors. It is not clear when epigenetic changes occur duringbreast cancer progression. However, methylation of the promoter was notdetected in both mortal, and immortal human mammary epithelial cell(HMEC) strains, as well as in normal microdissected breast epithelialcells. These results suggest that aberrant methylation of the RAR β2CpGisland may be a later event following immortalization. Treatment ofbreast cancer cells presenting with a methylated RAR β2, with thedemethylating agent 5-Aza-CdR, induced partial DNA demethylation andrestored RAR β gene expression. This evidence clearly indicates that DNAmethylation is at least a component contributing to RAR βdownregulation/loss.

EXAMPLE 5 Hypermethylation of HOXA5

The extent of methylation of the HOXA5-associated CpG islands in normalmammary epithelium, in breast cancer cell lines, and in primary mammarytumors was examined.

Tissue preparations and cells Freshly excised primary breast carcinomasor mammoplasty specimens were minced fine with razor blades and digestedwith 0.15% collagenase A and 0.5% dispase II (Boehringer Mannheim)prepared in RPMI 1640 medium. The cell clumps were separated from thelighter fibroblasts by gravity separation 3 times. The cell clumps werethen digested for 15′ with trypsin, washed, and immunostained withanti-cytokeratin-specific antibody (CAM 5.2, Becton-Dickinson) to assessthe level of epithelial cell enrichment. The epithelial cells comprisedbetween 70-80% of the enriched cell population.

Frozen, surgically excised breast tumor samples were cryosectioned, andrepresentative sections were screened by a pathologist after stainingwith hematoxylin and eosin. Sections containing more than 70% carcinomacells were used for RNA and protein extractions directly. Breast cancercell lines and immortalized HMECs were obtained from ATCC (Rockville,Md.). Finite life span HMECs were obtained from Dr. Martha Stampfer,HMEC strain 9F1403 was obtained from Clonetics.

Methylation specific PCR (MSP) and sodium bisulfite DNA sequencing Oneμg of genomic DNA was treated with sodium bisulfite²¹ and was analyzedfor MSP using primer sets specific for methylated DNA:5′-TTTAGCGGTGGCGTTCG-3′ (sense; SEQ ID NO:69) and5′-ATACGACTTCGAATCACGTA-3′ (antisense; SEQ ID NO:70), and primersspecific for unmethylated DNA: 5′-TTGGTGAAGTTGGGTG-3′ (sense; SEQ IDNO:71), and 5′-AATACAACTTCAAATCACATAC-3′(antisense; SEQ ID NO:72) whichyielded products of 183 and 213 bp respectively. Sodium bisulfitetreated DNA was used to PCR-amplify the HOXA5 promoter region −97 to−303 bp, using the primers 5′-ATTTTGTTATAATGGGTTGTAAT-3′ (sense; SEQ IDNO:73) and 5′-AACATATACTTAATTCCCTCC-3′ (antisense; SEQ ID NO:74). Theproduct was purified using a Qiagen PCR purification kit (Qiagen Corp.)and was sequenced using the sense primer with an ABI automatedfluorescent sequencer according to the manufacturer's instructions.

Treatment of cells with 5′-aza-2′-deoxycytidine (5-aza-dC) MDA-MB-231breast cancer cells were treated with 0.75 μM 5-aza-dC (Sigma), andcollected at 0, 3 and 5 days later. RT-PCR was performed using primers:(sense; SEQ ID NO:75) 5′-TCATTTTGCGGTCGCTATCC-3′ and (antisense; SEQ IDNO:76) 5′-GCCGGCTGGCTGTACCTG-3′.

Immunoblot Analysis Proteins were visualized by Western analysis and 10%SDS-PAGE. The primary antibodies [anti-HOXA5 (HOXA5-2, BABCO), anti-p53(AB-6, Oncogene Science), or anti-β-actin (AC-15, Sigma), anti-p21(15091A, Pharmingen), anti-Mdm2 (65101A, Pharmingen), anti-PARP (AB-2,Oncogene Sciences), anti-dynein (Zymed) and anti- Na+, K+-ATPase (EdBenz, Johns Hopkins) (also used as loading controls, with actin)] wereused at 1:1000 dilution.

p53 inactivation by mutation is low (20%) in human breast cancer.Looking for other mechanisms that may account for loss of p53 functionin these tumors, the levels of p53 mRNA in breast cancer cell lines andin primary tumors was examined. p53 mRNA levels were 5-10 fold lower intumor cells than in normal breast epithelium. A subsequent study lookedfor a consensus protein binding sites in the p53 promoter (Reisman, etal. Proc. Natl. Acad. of Sci. USA 85:5146-5150 (1988)), including thoseof HOX proteins which are known to function as transcription factors(Deschamps, et al. Crit. Rev. Oncog. 3:117-173 (1992); Scott, Nat.Genetics 15:117-118 (1997)). Selected HOX genes are differentiallyexpressed in neoplasms of a number of tissues, but their functionalrelationship to the neoplastic phenotype remains to be elucidated. Sixputative HOX-core binding sequences (ATTA) were identified within the2.4 kb human p53 promoter. Of a number of HOX genes examined in breasttumor cells and control breast epithelium, HOXA5 mRNA levels weredrastically reduced in breast cancer cells. In fact, there was a tightcorrelation between p53 and HOXA5 mRNA levels in the ten cell linestested for both genes, with a correlation coefficient r=0.942. No suchdecreased expression was observed for HOXA10, B3, B7, or C8 mRNAs.

To test for σ causal relationship between the decreased expression ofp53 and HOXA5 mRNAs, ZR75.1 breast cancer cells or SAOS2 osteosarcomacells were co-transfected with the −356 bp or the −2.4 kb human p53promoter-Luciferase reporter together with HOX expression plasmids.HOXA5 transactivated the p53 promoter-dependent reporter activity up to25-fold in ZR75.1 cells and up to 7-fold in SAOS2 cells. This effect wasnot seen with other homeotic genes HOXB4, HOXB5 and HOXB7.

Positive regulation of transcription by HoxA5 was observed with themouse p53 promoter as well. A single putative Hox-binding sequence(located at nts −204 to −201) was identified in the upstream regulatoryregion of the murine p53 gene. SAOS2 cells were cotransfected with a−320 bp mouse p53 promoter fused to the CAT gene, together withexpression plasmids encoding full-length murine HoxA5, HoxA7, or HoxC8proteins. Similar to human HOXA5, a 15- to 20-fold increase in CATactivity in the SAOS2 cells cotransfected with the HoxA5 expressionplasmid was observed, but no significant effect of HoxA7 or HoxC8. Theseresults suggest that expression from the mouse p53 promoter isspecifically stimulated by HoxA5. To define the sequence requirementsfor the transactivation function, a deletion construct of thep53-promoter CAT construct was tested in cotransfection assays with thefull-length HoxA5 expression plasmid. A deletion to −153 bp in thepromoter region of the p53-CAT construct eliminated stimulation of CATactivity by the effector plasmid. A truncated HoxA5 protein termedpCMVΔHoxA5, lacking the homeodomain, was completely inactive in theseexperiments. Finally a “TT” to “GG” mutation in the core-binding site(−320 mp53MutCAT) that abolished DNA/protein complex formation in cellextracts (see below), completely abrogated transactivation of the CATreporter gene by HoxA5.

Direct binding of HoxA5 to the ATTA-containing site in the p53 promoter(positions −204 to −201) was demonstrated by electrophoretic mobilityshift (EMSA) and supershift assays. A band was observed in cell extractsfrom HoxA5 transfected cells, but not in extracts from control cells.This band was competed out by an excess of unlabeled oligonucleotide butnot by an oligonucleotide with an unrelated sequence. No protein/DNAcomplex was observed in extracts mixed with an oligonucleotide primerwhich carries two mutations (TT to GG) in the core binding site.Finally, HOXA5 antibodies, but not pre-immune serum, caused a supershiftof the bound HOXA5 protein/oligonucleotide complex. This supershift wasabrogated by pre-incubation with excess antibody (antigen depletion).Similar shift patterns were observed in extracts of RKO cellstransfected with the effector plasmid. These results indicate that theATTA-containing sequence in the mouse p53 promoter is indeed aHoxA5-binding sequence.

The above results suggest that HOXA5 may possess growth-suppressiveproperties through activation of p53 expression. To test thispossibility, breast cancer cells, MCF-7 and ZR75.1, which harborwildtype p53 genes, were transfected with the full length HOXA5 and theΔHOXA5 (homeodomain-deleted) expression plasmids and tested forcolony-forming ability. No surviving colonies were obtained fromHOXA5-transfected cells whereas those transfected with ΔHOXA5 and thevector control generated colonies with equal efficiency. To obtainstable cultures that could express HOXA5, clones of MCF-7 cells weregenerated in which the HOXA5 gene was placed under the control of anecdysone-inducible promoter. Within 3 hours after induction of HOXA5expression by the ecdysone analog, Ponasterone A (Pon A), the levels ofp53 mRNA rose by 2-fold. Western blotting showed that p53 and itsdownstream targets, p21 and Mdm2, as well as HOXA5 were reproduciblyinduced 2-5 fold following treatment with Pon A. Moreover, addition ofPon A resulted in cell shrinkage by 24 hours followed by significantcell death (80-90%) after 48 hours. Cell death occurred by apoptosisaccording to the following criteria: 1) cells shrank and formedcontractile bodies; 2) DNA laddering was observed; 3) poly (ADP-ribose)polymerase, a substrate for caspases, underwent cleavage by 12 hours;and 4) 70% of the cells showed micronucleus formation, membraneblebbing, and ghost cell features upon staining with acridine orange.This apoptosis was not accompanied by a detectable change in the levelsof Bax protein.

The results herein are consistent with the hypothesis that an increasein the level of HOXA5 in MCF-7 cells leads to an increase in p53 levels,which in turn results in apoptosis. As a further proof of this model,MCF-7 cells expressing the E6 gene of human papilloma virus, whentransfected with the HOXA5 expression vector, were fully able to formcolonies. Presumably, the induced p53 in these cells was sequestered byE6 protein and was unable to induce apoptosis. These results support theidea that HOXA5 induces apoptosis through a p53-dependent pathway inMCF-7 cells. This is the first demonstration of the involvement of a HOXprotein in apoptosis.

The hypothesis that HOXA5-induced apoptosis is mediated by p53 wastested as follows. The p53+/+ HCT 116 line of colon carcinoma cells andits p53−/− derivative clone 379.2 were transfected with HOXA5 and p53expression vectors. Expression of HOXA5 or p53 in the parental HCT116cells reduced the ability of the cells to form colonies. In contrast,HOXA5 and p53 expression led to different phenotypes in p53 null 379.2cells. Whereas expression of p53 in these cells abrogated colonyformation, expression of HOXA5 had no detectable effect. In theHOXA5-transfected cultures, stable colonies, expressing detectableamounts of HOXA5 protein, and of a size and number comparable to thevector control were observed. Thus, HOXA5 induces cell death only in thepresence of a wild-type p53 gene, adding further evidence that p53mediates HOXA5 activity. Conversely, cells lacking HOXA5 and p53 wouldbe unable to mount a normal response to treatments, such as DNA damage,that normally raise p53 levels by stabilizing the protein. To test thispossibility, the two tumor cell lines 21PT and 21MT, which have lowexpression of HOXA5 and p53 were treated with γ-radiation. No detectableincrease in p53 level in 21 PT and 21 MT was observed, while, asexpected, p53 was induced in MCF-7 cells.

These findings in cell culture experiments have in vivo correlates. Insixty-seven percent (20/30) of primary breast tumors, HOXA5 protein wasundetectable. Strikingly, concurrent loss of p53 expression was observedin the same tumors that lacked HOXA5. Among those tumors expressingHOXA5, one showed a band migrating faster than wild-type HOXA5 presentin the RKO cells. HOXA5 cDNAs from eleven p53-negative breast cancersamples and two finite life span human breast epithelial cell (HMEC)strains were sequenced. All HOXA5 coding regions were wild type, exceptthat of tumor #5 which contained a frameshift mutation (G insertion atcodon 204) that created a premature stop codon. There is a coupled lossof p53 and HOXA5 expression in primary breast carcinomas, possibly dueto lack of expression or mutational inactivation of HOXA5.

Seeking an explanation for the absence of the protein in the tumors,HOXA5 DNA of 20 HOXA5-negative and 5 HOXA5-positive primary tumors wassequenced. All contained the wild-type sequence except tumor #5, inwhich the insertion of G was again found and which contained nowild-type allele. In the absence of mutations, loss of HOXA5 may be aconsequence of a loss of upstream regulatory factors or may reflect somerepressive phenomenon such as methylation of the gene. Methylationspecific PCR (MSP) of sodium bisulfite-treated DNA showed that 16/20 ofthe tumors contained partially or completely methylated CpGs in theHOXA5 promoter region (ACCN No. AC004080). In contrast, this region wascompletely unmethylated in human mammary epithelial cells (HMEC) offinite life span, 184 and 9F1403, and in 4 immortalized HMECs, HBL100,MCF10A, 184B5 and 184A1. Nucleotide sequencing of the region −97 bp to−303 bp of the HOXA5 promoter, using sodium bisulfite-treated DNA fromHMEC 184, and cancer cell lines, MCF-7 and MDA-MB-231, showed thatmethylation correlated with silencing of gene expression. Expression ofHOXA5 mRNA could be re-initiated in MDA-MB-231 cells by treatment withthe DNA methyl transferase inhibitor, 5-aza-2′-deoxycytidine (5-aza-dC).These results are strong preliminary evidence that methylation of theHOXA5 promoter region may be responsible for silencing of geneexpression.

Unlike most tumor types, up to 80% of sporadic breast cancers do notcontain p53 mutations. These results suggest that the reduced p53 levelsin these tumors result from the absence of a positive regulator of p53mRNA synthesis. p53 normally functions as a tetramer, so even a smallreduction in the concentration of p53 monomers can greatly reduce theeffective concentration of tetramers. These results show for the firsttime that transfected HOXA5 upregulates both p53 promoter-reporterconstructs and endogenous p53 synthesis, leading to apoptosis. Finally,HOXA5 was detectable in only one-third of the primary tumors. In themajority of the remaining tumors, lack of HOXA5 expression stronglycorrelated with methylation of its promoter region, suggesting a causalrole for methylation in the silencing of HOXA5 gene expression.

In summary, these experiments show that HOXA5 is a positive regulator ofp53 transcription and function in cultured cells. The correlationobserved between HOXA5 and p53 levels in clinical breast cancerdemonstrates that loss of HOXA5 expression is an important step intumorogenesis.

EXAMPLE 6 Hypermethylation of NES-1

The extent of methylation of the NES-1-associated CpG islands in normalmammary epithelium, in breast cancer cell lines, and in primary mammarytumors was examined.

Cell Lines and Tissues The immortalized HMECs 184A1 (passage 15 and 99)were kindly provided by Dr. Martha Stampfer, and grown as decribed(http://www.lbl.gov/LBL-Programs/mrgs/review.html; incorporated byreference herein). Mammary organoids were prepared from reductionmammoplasty specimens of women with benign or no abnormalties in thebreast following collagenase digestion as described (Bergstraessar andWeitzman (1993) Cancer Res., 53:2644-2654). Primary breast tumor tissueswere obtained after surgical resection at the John Hopkins University,and stored frozen at −80° C. DNA was extracted by standard methods ( ).RNA was extracted with Triazol.

Methylation-specific PCR (MSP) One μg genomic DNA was treated withsodium bisulfite as described in Herman et al. (supra), and was analyzedby MSP using primer sets located within the third exon of Nes 1 gene.Primers specific for unmethylated DNA were 5′-TTGTAGAGGTGGTGTTGTTT-3′(sense; SEQ ID NO:77) and 5′-TTGTAGAGGTGGTGTTGTTT-3′ (antisense; SEQ IDNO:78) and yielded a 128 base-pairs PCR product. Primers specific formethylated DNA were 5′-TTCGAAGTTTATGGCGTTTC-3′ (sense; SEQ ID NO:79) and5′-TTATTTCCGCAATACGCGAC-3′ (antisense; SEQ ID NO:80) and yielded a 137base-pairs PCR product. The PCR conditions were as follows: 1 cycle of95° C. for 5 min “hot start”, then addition of 1 u of Taq polymerase(RedTaq); 35 cycles of 95° C. for 30 s, 55° C. for 30 s and 72° C. for45 s; and 1 cycle of 72° C. for 5 min. The PCR products were resolved byelectrophoresis in a 2% agarose gel in 1× TBE buffer.

RT-PCR RNA was treated with RNAse-free DNAse (Boehringer-Mannheim) (0.51 u/ul) for 30 min. at 37° C., followed by heat inactivation at 65° C.for 10 min. RT reactions contained 2 μg DNAse treated RNA, 0.25 μg/μlpdN6 random primers (Pharmacia), 1× first strand buffer (GibcoBRL), 1 mMof each dNTP (Pharmacia), and 200 U MMLV-RT (GibcoBRL), and wereincubated for 1 h at 37° C. followed by heat inactivation at 75° C. for5 min. PCR was performed using the primers 5′-ACCAGAGTTGGGTGCTGAC-3′(sense; SEQ ID NO:81) and 5′-ACCTGGCACTGGTCTCCG-3′ (antisense; SEQ IDNO:82) for Nes1. A “housekeeping” ribosomal protein gene 36B4 wasco-amplified as an internal control, using primers5′-GATTGGCTACCCAACTGTTGCA-3′(sense; SEQ ID NO:83) and5′-CAGGGGCAGCAGCCACAAAGGC-3′ (antisense; SEQ ID NO:84). The 25 μlreactions contained 1× buffer (1:10 of 10× PCR buffer BRL#, 1.2 mMMgSO4, 0.2 mM of each dNTP) and 100 nM of each primer. The PCRconditions were: 1 cycle of 94° C. for 1 min “hot start” then additionof 1 u of Taq polymerase (RedTaq); 1 cycle of 94° C. for 2 min; 35cycles of: 94° C. for 30 sec, 55° C. for 30 sec, 72° C. for 45 sec andfinally 5 min. The PCR samples were resolved by electrophoresis on a 2%agarose gel in 1× TBE buffer.

NES-1 expression was observed in mammary organoids and HMEC's frommammoplasty specimens of normal and benign disease breast. In finitelife span HMEC primary breast carcinomas analyzed by RT-PCR, NES-1expression was observed in seven of eleven samples. MSP analysis for aCpG-rich island at NES-1 third exon in the same samples showedmethylated sequences in samples that showed NES-1 expression andunmethylated sequences in samples without NES-1 expression. MethylatedNES-1 is absent in normal tissue.

EXAMPLE 7

In earlier examples use of methylation-specific polymerase chainreaction (PCR) technology (MSP) for detection of the promotermethylation status of human cyclin D2, retinoic acid receptor beta(RARP), and Twist genes (called “direct MSP”) is described. These genesare essentially unmethylated in normal tissue, but high levels ofmethylation were found in carcinoma. The present example illustrates abroad study of ductal and lobular carcinoma employing two additionalmarkers, RASSF1A, and Hin-1 genes, in order to achieve the goal ofdetection of 100% of breast carcinomas. Results of this study show that100% of invasive ductal carcinoma patients can be detected using thecombination of Cyclin D2, RARβ, Twist, and RASSF1A markers (N=27patient). In addition 100% of invasive lobular carcinoma patients can bedetected using the combination of Cyclin D2, RARβ, Twist, and Hin-1markers (N=19 patients). In the study of 129 patients, the incidence (%)of patients detected with methylation of each of these genes in breastcarcinoma is as indicated in Table 3 below. TABLE 3 Cyc D2 RAR betaTwist RASSF1A Hin1 19 25 20 62 53 LCIS, in situ 35 20 20 85 79 Lobularcarcinoma, invasive 28 33 20 80 75 Grade 1 DCIS, in situ 21 50 23 50 58Grade 2 DCIS, in situ 42 47 42 78 63 Grade 3 DCIS, in situ 54 30 47 6659 Ductal carcinoma, invasive

Thus, the direct MSP technology provides a mechanism for detection ofmost human breast cancer by molecular methods.

Potential problems limiting such analyses are mainly the small amount ofDNA that is available under certain circumstances (e.g. in ductallavage, where fluid and cells are obtained from the breast duct) and theneed to enhance detection of trace amounts of methylated tumor (e.g. inanalyses of blood for circulating tumor DNA, diluted by the presence ofa vast excess of unmethylated DNA from blood cells). These problems havenow been overcome by development a new technology called multiplex MSP.The procedure for multiplex MSP is basically the following three steps:

-   -   1. DNA is isolated and treated with sodium bisulfite, as in        direct MSP.    -   2. PCR reaction #1 is performed using 2 μl DNA (□0.1 μg) in the        presence of 5 pairs of primers that will specifically amplify        Cyclin D2, RARβ, Twist, RASSF1A, and Hin-1 in the same tube.        These primers bind DNA whether or not it is methylated and they        bind outside the region that is amplified in PCR reaction #2.    -   3. PCR reaction #2 is performed using 1 μl diluted PCR-derived        DNA from the first PCR reaction. As in direct MSP, one pair of        primers is used per tube that will amplify one gene (either        Cyclin D2, RARβ, Twist, RASSF1A, or Hin-1) and the primers are        methylation status-specific. Thus two tubes are run per test        (patient sample) in PCR reaction #2, each for detection of        either unmethylated or methylated DNA respectively. In this        reaction PCR-derived DNA is diluted between 10¹ and 10⁷ fold        (See FIG. 11).

In more detailed terms, multiplex methylation-specific PCR wasaccomplished by performing two sequential PCR reactions. The first PCRreaction used 5 pairs of gene-specific external primers to co-amplifyCyclin D2, RARβ, Twist, RASSF1A, and Hin-1. The external primer pairshybridized to sequences outside the region covered by the second PCRreaction. External primers do not contain CpG sequences, thus DNAamplification was independent of methylation status of the genome. Thesecond PCR reaction used 1 pair of gene-specific internal primers toamplifiy DNA. Unlike the first PCR reaction, for the second PCR reactionprimers were methylation status-specific. All primers recognized onlysodium bisulfite treated DNA (data not shown). The primer sequencesutilized are shown in Table 4 for each gene.

For the first PCR reaction 2 μl sodium bisulfite-treated DNA was addedto a reaction mixture containing 166 mM (NH₄)₂S0₄, 670 mM Tris, pH 8.8,67 mM MgCl₂, 100 mM β-mercaptoethanol, 1% DMSO and 4 μg/ml of eachexternal primer, in a final volume of 25 μl. The reaction was overlaidwith 2 drops oil in a 500 μl eppendorf tube. Samples were incubated at95° C. for 5 min, and then 35 cycles of 95° C. for 30 sec, 56° C. for 30sec, and 72° C. for 45 sec. The final extension was performed at 72° C.for 5 min. For the second PCR reaction, 1 μl of the first PCR reaction(diluted 1:10²-1:10⁶) was added to the PCR reaction mix, as describedabove, which in addition contained 4 μg/ml of each of two internalprimers (forward and reverse). External primers were not added.Reactions to detect methylated and unmethylated genome were carried outin separate reaction tubes, in 8-well strip tubes covered with 2 dropsof oil/well. PCR reaction conditions were identical to the firstreaction.

Using this technique, it was determined that multiplex MSP greatlyenhances the amount of DNA available for analyses of markers of tumormethylation. The test capacity for direct MSP if ˜1 μg starting DNA isused enables evaluation of 5 genes in duplicate. By comparison, if ˜0.1μg of starting DNA is used in multiplex MSP, a panel of 5 genes can beevaluated in 25 replicate tests, and there is the potential that 10panels of 5 genes in replicates of 25 tests could be evaluated from ˜1μg starting DNA. This would be true if the PCR reaction DNA wasconservatively diluted only 10¹ fold, and we have observed that it maybe possible to dilute it much higher (i.e. 10⁵-10⁶ fold) to furtherenhance the availability of sample DNA.

Multiplex MSP was found to be highly specific, demonstrating concordancewith direct MSP analyses of samples obtained from normal human whiteblood cells (WBC), breast cancer cell lines, and primary breast tumors.Samples found unmethylated by direct MSP were unmethylated by multiplexMSP as well. Furthermore, higher sensitivity for detection of methylatedDNA was observed with multiplex MSP, as traces of methylated DNA weredetectable by multiplex MSP that were not detectable by direct MSP insome samples.

In these studies, Cyclin D2, ASSF1A and/or Twist were found to bemethylated (at least one marker) in 100% of invasive ductal carcinomasin a sample of 27 cell lines tested. Also gene promotor methylation wasfound in invasive lobular carcinoma cells as follows: RASSF1A=85%(n=20); HIN-1=79% (n=19); Twist=20% (n=20); RARβ=20% (n=20); andCyclinD2=35% (n=20). Gene promotor methylation was found in invasiveductal carcinoma cells as follows: RASSF1A=66% (n=20); HIN-1=59% (n=19);Twist=47% (n=20); RARβ=30% (n=20); and CyclinD2=54% (n=20). incidence ofvarious combinations of cyclin D2, RARβ, Twist, TASSF1A and HIN-1 ininvasive ductal carcinoma in a study of breast cancer cell lines (n=27)was also determined using Multiplex methylation-specific PCR. Thecombination of cyclin D2, RARβ and Twist occurred in 89% of the samples;the combination of cyclin D2, RARβ, Twist and RASSF1A occurred in 100%of the samples; and the combination of Cyclin D2, RARβ, Twist, and Hin-1occurred in 93% of the samples tested. The combination of RASSF1A andHIN-1 detected invasive lobular carcinoma with 95% accuracy. Thesestudies show that RASSF1A and HIN-1 are preferred markers for evaluatinga subject having or suspected of having early stage tumorogenesis ofbreast tissue and that a Multiplex methylation-specific PCR assayutilizing the five markers RASSF1A, Twist and Cyclin D2 will provide anaccuracy of 100% detection of invasive ductal carcinoma.

In conclusion, the multiplex MSP technology can greatly enhance thedetection of trace amounts of methylated DNA from patient samples, in amanner which is highly specific. Multiplex MSP can also greatly increasethe amount of DNA available for analyses of a wider number of markers oftumor methylation than can presently be analyzed by direct PCR. Thistechnology could allow for analyses of up to 50 genes (10 panels of 5genes) from the same amount of starting material that can maximally beused to analyze 5 genes using direct MSP.

Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims. TABLE 4 SEQ ID Sense/ NO: Gene antisense 1 WT Sense5′-GCGGCGCAGTTCCCCAACCA-3′ nucleotides 882- 901 2 WT antisense5′-ATGGTTTCTCACCAGTGTGCTT-3′ nucleotides 1416- 1437 3 WT Sense5′-GCATCTGAAACCAGTGAGAA-3′ nucleotides 1320-1339 4 WT antisense5′-TTTCTCTGATGCATGTTG-3′ nucleotides 1685- 1702 5 WT Sense5′-GATTGGCTACCCAACTGTTGCA-3′ 6 WT antisense 5′-CAGGGGCAGCAGCCACAAAGGC-3′7 WT sense 5′-TTTGGGTTAAGTTAGGCGTCGTCG-3′ 8 WT antisense5′-ACACTACTCCTCGTACGACTCCG-3′ 9 WT sense 5′-TTTGGGTTAAGTTAGGTGTTGTTG-3′10 WT antisense 5′-ACACTACTCCTCATACAACTCCA-3′ 11 WT sense5′-CGTCGGGTGAAGGCGGGTAAT-3′ 12 WT antisense 5′-CGAACCCGAACCTACGAAACC-3′13 WT sense 5′-TGTTGGGTGAAGGTGGGTAAT-3′ 14 WT antisense5′-CAAACCCAAACCTACAAAACC-3′ 15 cyclin D2 sense5′-CATGGAGCTGCTGTGCCACG-3′ 16 cyclin D2 antisense5′-CCGACCTACCTCCAGCATCC-3′ 17 cyclin D1 sense 5′-AGCCATGGAACACCAGCTC-3′18 cyclin D1 antisense 5′-GCACCTCCAGCATCCAGGT-3′ 19 cyclin D2 sense5′-GATTGGCTAC CCAACTGTTGCA-3′ 20 cyclin D2 antisense5′-CAGGGGCAGCAGCCACAAAGGC-3′ 21 cyclin D2 sense5′-GTTATGTTATGTTTGTTGTATG-3′ unmethylated 22 cyclin D2 antisense5′-GTTATGTTATGTTTGTTGTATG-3′ unmethylated 23 cyclin D2 sense5′-TACGTGTTAGGGTCGATCG-3′ methylated 24 cyclin D2 antisense5′-CGAAATATCTACGCTAAACG-3′ methylated 129 cyclin D2 sense5′-TATTTTTTGTAAAGATAGTTTTGAT-3′ External 130 cyclin D2 antisense5′-TACAACTTTCTAAAAATAACCC-3′ External 25 14.3.3 sense5′-ACAGGGGAACTTTATTGAGAGG-3′ A 375 bp σ-specific sigma probe 26 14.3.3antisense 5′-AAGGGCTCCGTGGAGAGGG-3′ (SEQ ID NO:26) sigma 27 14.3.3 sense5′-GAGGAGTGTCCCGCCTTGTGG-3′ A TG repeat sigma sequence in the 3′UTR of σ28 14.3.3 antisense 5′-GTCTCGGTCTTGCACTGGC3′ sigma 29 14.3.3 sense5′-GTGTGTCCCCAGAGCCATGG-3′ A 1.2 kb PCR sigma product, encompassing theentire σ coding sequence, was generated using two primers 30 14.3.3antisense 5′-GTCTCGGTCTTGCACTGGCG-3′ (antisense; sigma SEQ ID NO:30 3114.3.3 antisense 5′-CACCTTCTCCCGGTACTCACG-3′ entire σ sigma codingsequence: 32 14.3.3 sense 5′-GAGCTCTCCTGCGAAGAG-3′ entire σ sigma codingsequence: 33 14.3.3 sense 5′-GAGGAGGCCATCCTC TCTGGC-3′ entire σ sigmacoding sequence: 34 14.3.3 antisense 5′-TCCACAGTGTCAGGTTGTCTCG-3′ entireσ sigma coding sequence: 35 14.3.3 sense 5′-GAGAGAGTTAGTTTGATTTAGAAG-3′start at nt 8641 sigma, generates a first exon 474 bp PCR product 3614.3.3 antisense 5′-CTT ACTAATATCCATAACCTCC-3′ (antisense sigma primerwith start at nt 9114; 37 14.3.3 sense 5′-TGGTAGTTTTTATGAAAGGCGTC-3′methylated sigma DNA 38 14.3.3 antisense 5′-CCTCTAACCGCCCACCACG-3′ sigma39 14.3.3 sense 5′-ATGGTAGTTTTTATGAAAGGTGTT-3′ unmethylated sigma DNA 4014.3.3 antisense 5′-CCCTCTAACCACCCACCACA-3′ sigma 41 14.3.3 sense5′-GTGTGTCCCCAGAGCCATGG-3′ PCR was sigma performed using the σ- specificprimers 42 14.3.3 antisense 5′-ACCTTCTCCCGGTACTCACG-3′ sigma 43 RARβsense 5′-AGA GTT TGA TGG AGTTGG GTG GAG-3′ 227 bp probe was amplified 44RARβ antisense 5′-CAT TCG GTT TGGGTC AAT CCA CTG-3′ 45 RARβ sense5′-CAGCCCGGGTAGGGTTCACC-3′ W3 46 RARβ antisense5′-CCGGATCCTACCCCGACGG-3′ W3 47 RARβ sense 5′-CCGAGAACGCGAGCGATCC-3′ W448 RARβ anti- 5′-GGCCAATCCAGCCGGGGCG-3′ W4 sense 49 RARβ sense 5′-GTGGGT GTA GGT GGA ATA TT-3′ unmethylated DNA were as follows: U1 50 RARβantisense 5′-AAC AAA CAC ACA AAC CAA CA-3′ U1 51 RARβ sense 5′-TGT GAGTTA GGA GTA GTG TTTT-3′ U2 52 RARβ antisense 5′-TTC AAT AAA CCC TACCCA-3′ U2 53 RARβ sense 5′-TTA GTA GTT TGG GTA GGGTTT ATT-3′ U3 54 RARβantisense 5′-CCA AAT CCT ACC CCAACA-3′ U3 55 RARβ sense 5′-GAT GTT GAGAAT GTGAGT GAT TT-3′ U4 56 RARβ antisense 5′-AAC CAA TCC AACCAA AAC A-3′U4 57 RARβ sense 5′-AGC GGGCGT AGG CGG AAT ATC-3′ methylated M1 58 RARβantisense 5′-CAACGA ACG CAC AAA CCG ACG-3′ M1 59 RARβRAR sense 5′-CGTGAG TTA GGA GTA GCG TTT C-3′ M2 β 60 RARβ antisense 5′-CTT TCG ATA AACCCT ACC CG-3′ M2 61 RARβ sense 5′-GGT TAG TAG TTC GGG TAG GGTTTA TC- M33′ 62 RARβ antisense 5′-CCG AAT CCT ACC CCGACG-3′ M3 63 RARβ sense5′-GTC GAG AAC GCG AGCGAT TC-3′ M4 64 RARβ antisense 5′-CGA CCA ATC CAACCGAAA CG-3′ M4 65 RARβ sense 5′-GAC TGT ATG GAT GTTCTG TCA G-3′ RT± PCR exon 5 66 RARβ antisense 5′-ATT TGTCCT GGC AGA CGA AGC A-3′ exon 6133 RARβ sense 5′-GTAGGAGGGTTTATTT TTTGTT-3′ External 134 RARβ antisense5′-AATTACATTTTCCAAACTTACTC-3′ External 135 RARβ sense5′-GGATTGGGATGTTGAGAATGT-3′ Methylated 136 RARβ antisense5′-AACCAATCCAACCAAAACAA-3′ Methylated 92 RARβ sense5′-GGATTGGGATGTTGAGAATGT-3′ Unmethylated 93 RARβ antisense5′-CAACCAATCCAACCAAAACAA-3′ Unmethylated 67 Actin sense 5′-ACC ATG GATGAT GAT ATCG-3′ RT ± PCR 68 Actin antisense 5′-ACA TGG CTG GGG TGTTGAAG-3′ 69 HOXA5 sense 5′-TTTAGCGGTGGCGTTCG-3′ methylated DNA 70 HOXA5antisense 5′-ATACGACTTCGAATCACGTA-3′ 71 HOXA5 sense5′-TTGGTGAAGTTGGGTG-3′ unmethylated 72 HOXA5 antisense5′-AATACAACTTCAAATCACATAC-3′ 73 HOXA5 sense 5′-ATTTTGTTATAATGGGTTGTAAT3′74 HOXA5 antisense 5′-AACATATACTTAATTCCCTCC-3′ 75 HOXA5 sense5′-TCATTTTGCGGTCGCTATCC-3′ RT-PCR 76 HOXA5 antisense5′-GCCGGCTGGCTGTACCTG-3′ 77 NES-1 sense 5′-TTGTAGAGGTGGTGTTGTTT-3′unmethylated 78 NES-1 antisense 5′-CACACAATAAAACAAAAAACCA-3′ 79 NES-1sense 5′-TTCGAAGTTTATGGCGTTTC-3′ Methylated 80 NES-1 antisense5′-TTATTTCCGCAATACGCGAC-3′ 81 NES-1 sense 5′-ACCAGAGTTGGGTGCTGAC-3′ 82NES-1 antisense 5′-ACCTGGCACTGGTCTCCG-3′ 83 36B4 sense5′-GATTGGCTACCCAACTGTTGCA-3′ 84 36B4 antisense5′-CAGGGGCAGCAGCCACAAAGGC-3′ 85 Estrogen sense 5′-G GGTGTTTTTGAGATTGTTGG-3 Unmethylated Receptor 86 5′-TG AGTTGTGATG GGTTTTGG-3 87antisense 5′-CCAAAACC CATCACAACT CA-3 88 sense 5′-AGAGTAGGCG GCGAGCGT-3Methylated 89 5′-CGGGAAAAG TACGTGTTCG T-3 90 antisense 5′-A CGAACACGTACTTTTCCCG-3 107 Twist sense 5′-T TTCGGATGGG GTTGTTCATC-3 Methylated 108Twist antisense 5′-AAACGAC CTAACCCGAA CG-3 Methylated 109 Twist sense5′-TT TGGATGGGGT TGTTATTGT-3 Unmethylated 110 Twist antisense 5′-CCTAACCCAAA CAACCAACC-3 Unmethylated 133 Twist sense5′-GAGATGAGATATTATTTATTGTG-3 External 134 Twist antisense5′-AACAACAATATCATTAACCTAAC-3 External 111 HIN-1 sense5′-AGGGAAGtTTTTTTtATTTGGTT-3 112 HIN-1 antisense5′-GTGGTTTTGTTTTGTATGTTTTGGTG-3 113 HIN-1 antisense5′-CACCGAAACATACAAAACAAAACCAC-3 114 HIN-1 sense5′-GTTTGTTAAGAGGAAGTTTT-3 External 115 HIN-1 antisense5′-CACCGAAACATACAAAACAAACCAC-3 External 116 HIN-1 sense5′-GGTACGGGTTTTTTACGGTTCGTC-3 Methylated 117 HIN-1 antisense5′-AACTTCTTATACCCGATCCTCG-3 Methylated 118 HIN-1 sense5′-GGTATGGGTTTTTTATGGTTTGTT-3 Unmethylated 119 HIN-1 antisense5′-CAAAACTTCTTATACCCAATCCTCA-3 Unmethylated 122 RASSF1A sense5′-GGGAGTTTGAGTTTATTGAGT-3 External 123 RASSF1A antisense5′-ACCCCTTAACTACCCCTTC-3 External 124 RASSF1A sense 5′-GTTGGTATTC-3Methylated 125 RASSF1A sense 5′-GTTGGGCGC-3 Methylated 126 RASSF1Aantisense 5′-GCACCACGTATACGTAACG-3 Methylated 127 RASSF1A sense5′-GGTTGTATTTGGTTGGAGTG-3 Unmethylated 128 RASSF1A antisense5′-CTACAAACCTTTACACACAACA-3 Unmethylated

TABLE 5 Multiplex Is Highly Specific Concordance Observed Between DirectPCR and Multiplex PCR in Human Primary Breast Tumor Analyses Cyclin D2RARbeta Twist RASSF1A Hin-1 Tumor Direct Multi Diln Direct Multi DilnDirect Multi Diln Direct Multi Diln Direct Multi Diln 7157 U U 3 U U 4 UU 2 U U 4 U U 3 231 M M 2 M M 4 M M 2 M M 4 M M 2 7103 U U 3 U U 4 U U 1U/M U/M 4 M M 3 7107 U/Mw U/Mw 3 U/M U/M 4 U/M U/M 1 U/M U/M 4 M M 37109 U/M U/M 3 U/M U/M 4 U/M U/M 1 U U 4 U/M U/M 3 7140 U U 3 U U 4 U U1 U U 4 U U 3

TABLE 6 Multiplex Is Highly Specific Concordance Observed Between DirectPCR and Multiplex PCR in Human WBC DNA Analyses Cyclin D2 RARbeta TwistRASSF1A Hin-1 WBC Direct Multi Diln Direct Multi Diln Direct Multi DilnDirect Multi Diln Direct Multi Diln 7157 U U 5 U U 6 U U 6 U U 6 U U 57160 U U 3 U U 5 U U 2 U U 5 U U 3 7163 U U 5 U U 6 U U 6 U U 6 U U 57164 U U 5 U U 6 U U 6 U U 6 U U 5 H20 NR NR 1 NR NR 1 NR NR 0 NR NR 1NR NR 1

TABLE 7 Multiplex Is Highly Sensitive Methylated Signals Not Observed byDirect PCR are Revealed by Multiplex PCR in Human Breast CA Cell LineAnalyses Cyclin D2 RARbeta, Twist RASSF1A Hin-1 Cell Lines Direct MultiDiln Direct Multi Diln Direct Multi Diln Direct Multi Diln Direct MultiDiln 7160 U U 4 U U 4 U U/M 2 U U 4 U U 4  231 M M 4 M M 4 M M 2 M M 4 MM 4 MCF-7 U/M U/M 4 M M 4 M/Uw M 2 M M 4 Mw M 4 MCF-10A M M/Uw 4 U/M U/M4 U U/Mw 2 M M 4 U/M U/M 4 HBL100 M U/M 4 U/Mw U/M 4 U U/Mw 2 M U/M 4M/Uw M 4 ZR7 5-1 U U/Mw 4 M M/Uw 4 U/M U/M 2 M M 4 M M 4

1. A method detecting primary breast cancer in a subject comprisingdetermining the state of methylation of one or more CpG islands in thepromoter of HIN-1 nucleic acids isolated from a sample comprising blood,plasma, lymph, duct cells, ductal lavage fluid, nipple aspiration fluid,breast tissue, lymph nodes, bone marrow, or a combination thereof of thesubject, wherein a state of hypermethylation of one or more CpG islandsin the promoter of HIN-1 nucleic acids as compared with the state ofmethylation of one or more CpG islands in the promoter of HIN-1 nucleicacids in comparable samples obtained from a normal subjects isindicative of primary breast cancer in the subject. 2-3. (canceled) 4.The method of claim 1, wherein the state of methylation of the nucleicacids is determined simultaneously. 5-8. (canceled)
 9. The method ofclaim 1, wherein the sample comprises duct cells obtained by a procedureselected from ductal lavage, sentinel node biopsy, fine needle aspirate,routine operative breast endoscopy, nipple aspiration and core biopsy.10. (canceled)
 11. The method of claim 1, wherein determining the stateof methylation comprises amplifying the nucleic acid by means of atleast one sense primer and at least one antisense primer thatdistinguishes between methylated and unmethylated nucleic acids.
 12. Themethod of claim 11, wherein the primers hybridize with targetpolynucleotide sequences selected from SEQ ID NO:25-36, 41-48, andcombinations thereof.
 13. The method of claim 11, wherein the primersare selected from SEQ ID NO:21-24, 37-40, and combinations thereof. 14.The method of claim 1, further comprising contacting the nucleic acidwith a methylation-sensitive restriction endonuclease.
 15. The method ofclaim 14, wherein the methylation-sensitive restriction endonuclease isselected from the group consisting of MspI, HpaII, BssHII, BstUI andNotI.
 16. A method of determining a predisposition to primary breastcancer in a subject comprising determining the state of methylation ofone or more CpG islands in the promoter of HIN-1 nucleic acids isolatedfrom a sample comprising blood, plasma, duct cells lymph, ductal ravagefluid, nipple aspiration fluid, breast tissue, lymph nodes bone marrow,or a combination thereof of the subject, wherein a state ofhypermethylation of the CpG islands in the promoter of HIN-1 nucleicacid(s) as compared with the state of methylation of comparable nucleicacid obtained from normal subjects is indicative of a predisposition toprimary breast cancer in the subject. 17-19. (canceled)
 20. The methodof claim 16, wherein the sample comprises duct cells obtained by aprocedure selected from the group consisting of ductal lavage, sentinelnode biopsy, fine needle aspirate, routine operative breast endoscopy,nipple aspiration and core biopsy.
 21. (canceled)
 22. The method ofclaim 16, wherein determining the state of methylation comprisesanplifying the nucleic acid(s) by means of at least one sense primer andat least one antisense primer that distinguishes between methylated andunmethylated nucleic acid.
 23. The method of claim 22, wherein thenucleic acids are amplified simultaneously.
 24. The method of claim 22,wherein the primers hybridize with target polynucleotide sequencesselected from SEQ ID NO:25-36, 41-48, and combinations thereof.
 25. Themethod of claim 24, wherein the primers are selected from SEQ IDNO:21-24, 37-40, and combinations thereof.
 26. The method of claim 16,further comprising contacting the nucleic acid with amethylation-sensitive restriction endonuclease.
 27. The method of claim26, wherein the methylation-sensitive restriction endonuclease isselected from the group consisting of MspI, HpaII, BssHII, BstUI andNotI.
 28. A method for diagnosing primary breast cancer in a subjectcomprising: (a) contacting a nucleic acid-containing specimen selectedfrom blood, plasma, lymph, duct cells, ductal lavage fluid, nippleaspiration fluid, breast tissue, lymph nodes bone marrow, or acombination thereof of the subject with an agent that provides adetermination of the methylation state of CpG islands in the promoter ofHIN-1 nucleic acids in the specimen, and (b) identifying the methylationstate of at least one CpG island in the promoter of HIN-1, wherein theCpG islands in the promoter of HIN-1 hypermethylated compared to themethylation state of the same region of the same nucleic acid in normalsubjects. 29-33. (canceled)
 34. The method of claim 28, wherein theagent is at least one sense primer and at least one antisense primerthat hybridize with a target sequence in the nucleic acid.
 35. Themethod of claim 34, wherein the primers hybridize with targetpolynucleotide sequences selected from SEQ ID NO:25-36, 41-48, andcombinations thereof.
 36. The method of claim 34, wherein the primersare selected from SEQ ID NO:21-24, 37-40, and combinations thereof.37-39. (canceled)
 40. The method of claim 34, wherein the method employsmultiplex methylation-specific PCR.
 41. The method of claim 40, whereinthe specimen comprises breast duct or ductal fluid. 42-44. (canceled)